Detection of glucose in solutions also containing an alpha-hydroxy acid or a beta-diketone

ABSTRACT

Compositions and methods for determining the presence or concentration of glucose in a sample which may also contain an alpha-hydroxy acid or a beta-diketone. The method uses a compound having at least two recognition elements for glucose, oriented such that the interaction between the compound and glucose is more stable than the interaction between the compound and the alpha-hydroxy acid or beta-diketone, such that the presence of the alpha-hydroxy acid or the beta-diketone does not substantially interfere with said determination.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of applicationSer. No. 10/029,184 filed Dec. 28, 2001, which is a continuation-in-partof application Ser. No. 09/754,217 filed Jan. 5, 2001 and claims thebenefit of application Serial No. 60/363,885 filed Mar. 14, 2002,application Serial No. 60/329,746 filed Oct. 18, 2001 and applicationSerial No. 60/269,887 filed Feb. 21, 2001.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to the detection of glucose insamples which may also contain potential interfering compounds, such asα-hydroxy acids or β-diketones.

[0005] 2. Description of the Related Art

[0006] The complexation of carbohydrates, including glucose, withphenylboronic acid has been known for a long time and the reversibilityof that interaction has served as a basis for the chromatographicseparation of sugars. Specifically, in 1959, Lorand and Edwards reportedassociation constants for aqueous associations of phenylboronic acidwith many saturated polyols; binding interactions ranged from very weak(e.g., ethylene glycol, K_(d)=360 mM) to moderately strong (e.g.,glucose, K_(d)=9.1 mM). See J. Yoon, et al., Bioorganic and MedicinalChemistry 1(4):267-71 (1993). The binding mechanism is believed to occurthrough bonding of adjacent hydroxyl groups on glucose to hydroxylgroups on a boronate moiety.

[0007] U.S. Pat. No. 5,503,770 (James, et al.) describes a fluorescentboronic acid-containing compound that emits fluorescence of a highintensity upon binding to saccharides, including glucose. Thefluorescent compound has a molecular structure comprising a fluorophore,at least one phenylboronic acid moiety and at least one amine-providingnitrogen atom where the nitrogen atom is disposed in the vicinity of thephenylboronic acid moiety so as to interact intramolecularly with theboronic acid. Such interaction thereby causes the compound to emitfluorescence upon saccharide binding. See also T. James, et al., J. Am.Chem. Soc. 117(35):8982-87 (1995).

[0008] Additionally, fluorescent sensors using an anthrylboronicacid-containing compound for detecting blood glucose are known in theart. For example, J. Yoon, et al., J. Am. Chem. Soc. 114:5874-5875(1992) describe that anthrylboronic acid can be used as a fluorescentchemosensor for signaling carbohydrate binding, including binding ofglucose and fructose.

[0009] Unfortunately, compounds which interact with glucose in themanner described above also have a tendency to interact with othercompounds having hydroxyl groups, thus reducing the specificity of aglucose assay, especially when assaying physiological samples which maycontain interfering amounts of lactate, acetoacetate, etc. For example,some diabetic patients also develop lactic acidosis, in which bloodlactate levels are greater than 5 mmol/liter. Thus, there remains agreat need for glucose assays which are relatively insensitive topotentially interfering hydroxyl compounds, such as lactate.

BRIEF SUMMARY OF THE INVENTION

[0010] In one aspect, the present invention is directed to a method fordetecting the presence or concentration of glucose in a sample which mayalso contain an α-hydroxy acid or a β-diketone, which comprises:

[0011] a) exposing the sample to a compound having at least tworecognition elements for glucose, oriented such that the interactionbetween the compound and glucose is more stable than the interactionbetween the compound and the α-hydroxy acid or β-diketone, said compoundalso containing a detectable moiety having a detectable quality thatchanges in a concentration-dependent manner when said compound isexposed to glucose in said sample; and

[0012] b) measuring any change in said detectable quality to therebydetermine the presence or concentration of glucose in said sample,wherein the presence of the α-hydroxy acid or the β-diketone does notsubstantially interfere with said determination.

[0013] In another aspect, the present invention is directed to acompound having the following structure

[0014] wherein:

[0015] R₁ and R₂ are the same or different and are selected from thefollowing: i) hydrogen; ii) a substituent to modify the pKa andhydrolytic stability of the R₈ moiety, iii) a detectable moiety, or iv)a linking group capable of attachment to a solid support or a polymericmatrix, said support or matrix optionally containing a detectablemoiety;

[0016] R₃ is hydrogen or a linking group capable of attachment to asolid support or a polymeric matrix, said support or matrix optionallycontaining a detectable moiety;

[0017] R₄ and R₅ are the same or different and are selected from thefollowing: i) hydrogen, ii) a substituent to modify the pKa andhydrolytic stability of the R₈ moiety, iii) a detectable moiety, or iv)a linking group capable of attachment to a solid support or a polymericmatrix, said support or matrix optionally containing a detectablemoiety;

[0018] each Z is independently carbon or nitrogen;

[0019] R₆ and R₇ are the same or different and are i) linking groupshaving from zero to ten contiguous or branched carbon and/orheteroatoms, or ii) a linking group capable of attachment to a solidsupport or a polymeric matrix, said support or matrix optionallycontaining a detectable moiety;

[0020] R is selected from the following: i) an aliphatic and/or aromaticspacer containing from 1 to 10 contiguous atoms selected from the groupconsisting of carbon, oxygen, nitrogen, sulfur and phosphorus, ii) adetectable moiety, or iii) a linking group capable of attachment to asolid support or a polymeric matrix, said support or matrix optionallycontaining a detectable moiety;

[0021] each R₈ is the same or different and is an optionally protectedmoiety which when unprotected is capable of interaction with the vicinaldiol groups present in glucose; and

[0022] R₉ and R₁₀ are the same or different, and are i) hydrogen, ii) adetectable moiety, iii) a group which is a) a linking group capable ofattachment to a solid support or a polymeric matrix, said support ormatrix optionally containing a detectable moiety, and/or b) includes afunctional group capable of altering the physical properties of thecompound;

[0023] with the proviso that the indicator compound contains at leastone detectable moiety associated therewith, either directly or as partof the solid support or polymeric matrix.

[0024] In another aspect, the present invention is directed to adetection system which comprises a compound described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 illustrates the normalized fluorescence emission (I/Io@420nm) of an indicator as described in Example 1.

[0026]FIG. 2 illustrates the normalized fluorescence emission (I/Io@428nm) of an indicator as described in Example 2.

[0027]FIG. 3 illustrates the normalized fluorescence emission (I/Io@428nm) of an indicator as described in Example 3.

[0028]FIG. 4 illustrates the normalized fluorescence emission (I/Io@427nm) of an indicator as described in Example 4.

[0029]FIG. 5 illustrates the normalized fluorescence emission (I/Io@540nm) of an indicator as described in Example 5.

[0030]FIG. 6 illustrates the absorbance spectra of an indicator asdescribed in Example 6.

[0031] FIGS. 7-8 illustrate the ratio of the absorbance (450 nm/530 nm)of an indicator as described in Example 6.

[0032]FIG. 9 illustrates the normalized fluorescence emission (I/I₀ at550 nm) of an indicator as described in Example 6.

[0033]FIG. 10 illustrates the fluorescence spectrum, in the absence ofglucose and in the presence of 100 mM glucose, of an indicator asdescribed in Example 6.

[0034]FIG. 11 illustrates the normalized fluorescence emission (I/I₀ at550 nm), in the presence of glucose and lactate, of an indicator asdescribed in Example 6.

[0035]FIG. 12 illustrates the normalized fluorescence emission (I/I₀ at525 nm) of an indicator exposed to glucose as described in Example 10.

[0036]FIG. 13 illustrates the normalized fluorescence emission (I/I₀ at530 nm) of an indicator exposed to lactate as described in Example 10.

[0037]FIG. 14 shows the relative fluorescence emission (I@430 nm) of anindicator exposed to glucose and lactate as described in Example 11.

[0038]FIG. 15 shows the relative fluorescence emission (I@430 nm) of anindicator exposed to glucose and lactate as described in Example 12.

[0039]FIG. 16 illustrates the fluorescence of an indicator exposed toglucose and lactate as described in Example 13.

DETAILED DESCRIPTION OF THE INVENTION

[0040] In one aspect, the present invention provides a way to detect thepresence or concentration of glucose in a sample which may also containinterfering compounds, such as α-hydroxy acids or β-diketones. Suchpotentially interfering compounds include lactate, acetoacetate,β-hydroxy butyric acid, etc.

[0041] The present invention is carried out using an indicator compoundwhich is capable of recognizing glucose in a sample, but which is lesslikely to recognize interfering compounds in the sample. The indicatorcompound has at least two recognition elements for glucose, orientedsuch that the interaction between the indicator compound and glucose ismore stable than the interaction between the indicator compound and theinterfering compounds.

[0042] Suitable recognition elements include moieties which are capableof a preferably reversible interaction with glucose, especially with thediol groups present in glucose. Several such recognition elements areknown, and preferably include boronic acid, boronate ion, arseniousacid, arsenite ion, telluric acid, tellurate ion, germanic acid,germanate ion, etc. Most preferred are recognition elements containingboron. It will be understood that until use, the recognition elementsmay be capped with a protecting group. Such groups are well known, andinclude neopentyl glycol, pinacol, etc. In certain embodiments, thecapped recognition element is decapped in the medium in which thecompound is to be used (see, e.g., Example 5).

[0043] The recognition elements are preferably spaced on the indicatorcompound a suitable distance from each other so as to allow at least twoof the recognition elements to interact with a glucose molecule,resulting in increased specificity. In general, the recognition elementsmay have a spacer of up to about 30 atoms between them. Preferably, therecognition elements are oriented such that they are capable of beingabout 6 Å apart when interacting with glucose.

[0044] The indicator compounds of the present invention have adetectable quality that changes in a concentration-dependent manner whenthe compound is exposed to a sample containing glucose. Many suchqualities are known and may be used in the present invention. Forexample, the indicator compound may include a luminescent (fluorescentor phosphorescent) or chemiluminescent moiety, an absorbance basedmoiety, etc. The indicator compound may include an energy donor moietyand an energy acceptor moiety, each spaced such that there is adetectable change when the indicator compound interacts with glucose.The indicator compound may include a fluorophore and a quencher,configured such that the fluorophore is quenched by the quencher whenglucose is absent. In that situation, when glucose is present, theindicator undergoes a configurational change which causes the quencherto move sufficiently distant from the fluorophore so that fluorescenceis emitted. Conversely, the fluorophore and quencher may be configuredsuch that in the absence of glucose, they are sufficiently separated andthe fluorophore emits fluorescence; upon interaction with glucose, thefluorophore and quencher are moved in sufficient proximity to causequenching. The configurational change concept is described in moredetail in our co-pending application Ser. No. 09/754,219, filed Jan. 5,2001, entitled “Detection of Analytes”, incorporated herein byreference.

[0045] Alternatively, the indicator may include a moiety such as afluorophore capable of interacting with the recognition element oranother moiety spatially disposed with respect to the recognitionelement such that in the absence of glucose, the fluorophore emitsfluorescence. Upon addition of glucose, the glucose competes with theinteraction between the fluorophore and the recognition element, or theinteraction between the fluorophore and the other moiety spatiallydisposed with respect to the recognition element, causing a reduction influorescence. An example of that concept is illustrated in Example 6. Itwill also be recognized that the indicator may be chosen such that thefluorophore emits no fluorescence, or a relatively low level offluorescence, when the fluorophore interacts with the recognitionelement or another moiety spatially disposed with respect to therecognition element in the absence of glucose. Upon addition of glucose,the glucose competes with the interaction between the fluorophore andthe recognition element, or the interaction between the fluorophore andthe other moiety spatially disposed with respect to the recognitionelement, causing an increase in fluorescence.

[0046] Other detectable moieties include those whose fluorescence isaffected by glucose interaction via photoinduced electron transfer orinductive effects. These include the lanthamide chelates disclosed incopending U.S. application Ser. No. 09/265,979 filed Mar. 11, 1999 (andpublished as PCT International Application WO 99/46600 on Sep. 16,1999), incorporated herein by reference; polyaromatic hydrocarbons andtheir derivatives; coumarins; BoDiPy; dansyl; catechols; etc. Anotherclass of moieties include those whose absorbance spectrum changes uponinteraction of the indicator compound with glucose, including AlizarinRed, etc. Another class of moieties include those whose fluorescence ismodulated by proximity effects, e.g., energy donor/acceptor pairs suchas dansyl/dabsyl, etc.

[0047] Preferably, the detectable quality is a detectable spectralchange, such as changes in absorptive characteristics (e.g.,absorbtivity and/or spectral shift), in fluorescent decay time(determined by time domain or frequency domain measurement), fluorescentintensity, fluorescent anisotropy or polarization; a spectral shift ofthe emission spectrum; a change in time-resolved anisotropy decay(determined by time domain or frequency domain measurement), etc.

[0048] The indicator compounds of the present invention, if soluble, maybe used directly in solution if so desired. On the other hand, if thedesired application so requires, the indicator compounds may beimmobilized (such as by mechanical entrapment or covalent or ionicattachment) onto or within an insoluble surface or matrix such as glass,plastic, polymeric materials, etc. When the indicator compound isentrapped within, for example, another polymer, the entrapping materialpreferably should be sufficiently permeable to glucose to allow suitableinteraction between glucose and the indicator compound.

[0049] If the indicator compounds are sparingly soluble or insoluble inwater, yet detection in an aqueous medium is desired, the indicatorcompound may be co-polymerized with a hydrophilic monomer to form ahydrophilic macromolecule as described in co-pending U.S. applicationSer. No. 09/632,624, filed Aug. 4, 2000, the contents of which areincorporated herein by reference.

[0050] Preferred indicator compounds have the following structure:

[0051] wherein:

[0052] R₁ and R₂ are the same or different and are selected from thefollowing: i) hydrogen; ii) a substituent to modify the pKa andhydrolytic stability of the R₈ moiety, iii) a detectable moiety, or iv)a linking group capable of attachment to a solid support or a polymericmatrix, said support or matrix optionally containing a detectablemoiety;

[0053] R₃ is hydrogen or a linking group capable of attachment to asolid support or a polymeric matrix, said support or matrix optionallycontaining a detectable moiety;

[0054] R₄ and R₅ are the same or different and are selected from thefollowing: i) hydrogen, ii) a substituent to modify the pKa andhydrolytic stability of the R₈ moiety, iii) a detectable moiety, or iv)a linking group capable of attachment to a solid support or a polymericmatrix, said support or matrix optionally containing a detectablemoiety;

[0055] each Z is independently carbon or nitrogen;

[0056] R₆ and R₇ are the same or different and are i) linking groupshaving from zero to ten contiguous or branched carbon and/orheteroatoms, or ii) a linking group capable of attachment to a solidsupport or a polymeric matrix, said support or matrix optionallycontaining a detectable moiety;

[0057] R is selected from the following: i) an aliphatic and/or aromaticspacer containing from 1 to 10 contiguous atoms selected from the groupconsisting of carbon, oxygen, nitrogen, sulfur and phosphorus, ii) adetectable moiety, or iii) a linking group capable of attachment to asolid support or a polymeric matrix, said support or matrix optionallycontaining a detectable moiety;

[0058] each R₈ is the same or different and is an optionally protectedmoiety which when unprotected is capable of interaction with the vicinaldiol groups present in glucose; and

[0059] R₉ and R₁₀ are the same or different, and are i) hydrogen, ii) adetectable moiety, iii) a group which is a) a linking group capable ofattachment to a solid support or a polymeric matrix, said support ormatrix optionally containing a detectable moiety, and/or b) includes afunctional group capable of altering the physical properties of thecompound;

[0060] with the proviso that the indicator compound contains at leastone detectable moiety associated therewith, either directly or as partof the solid support or polymeric matrix.

[0061] Suitable groups for modifying the pKa and hydrolytic stability ofthe R₈ moieties would be readily apparent to one of ordinary skill, andinclude groups such as halogen; nitro; amino; halogen substituted alkyl;optionally substituted carboxyl; acyl; keto; nitrile; amide; ester;alkoxy; etc.

[0062] Suitable linking groups for any substituent may include groupsfrom about 1 to about 20 contiguous atoms, which may be branched orsubstituted and which may include one or more heteroatoms, whichterminate in a functional group capable of further reaction orattachment to a polymer or support. Examples of suitable linking groupsinclude alkyl; aryl; acyl; polyamide; polyether; all optionallysubstituted, and combinations thereof.

[0063] R₉ and R₁₀ may further include functional groups capable ofaltering the physical properties of the compound, such as solubility,pKa, etc. For example, these include optionally substitutedcarboxylates, amino groups, quartenary ammonium groups, sulfonates, PEG,etc.

[0064] It will be understood that when any of the substituents is adetectable moiety, that could also include suitable linking groups whichlink the detectable moiety to the rest of the indicator compound.Suitable linking groups include those listed above. Suitable detectablemoieties include those defined above.

[0065] R₈ is preferably selected from the group consisting of boronicacid, boronate ion, arsenious acid, arsenite ion, telluric acid,tellurate ion, germanic acid, germanate ion, and combinations thereof.

[0066] It will also be understood from the above definition that thepresent compounds and detection systems may be in polymeric form. Thus,an integral compound (containing recognition elements and detectablemoiety) could be linked to an existing polymer, or the integral compoundin monomeric form could be polymerized or co-polymerized with anothersuitable monomer to form a polymer. Alternatively, two separatemonomeric components (e.g., one containing the recognition elements, andone containing a detectable moiety) could be co-polymerized so that theresulting polymer contains all necessary elements of the system (seeExample 6).

[0067] Many uses exist for the indicator compounds of the presentinvention, including uses as indicators in the fields of energy,medicine and agriculture. For example, the indicator compounds can beused to detect sub-levels or supra-levels of glucose in physiologicalbuffers or fluids, such as blood, plasma, serum, interstitial fluid,cerebrospinal fluid, urine, saliva, intraocular fluid, lymph, tears, orsweat, thus providing valuable information for diagnosing or monitoringsuch diseases as diabetes and adrenal insufficiency.

[0068] Medical/pharmaceutical production of glucose for humantherapeutic application requires monitoring and control.

[0069] Uses for the present invention in agriculture include detectinglevels of glucose in soybeans and other agricultural products. Glucosemust be carefully monitored in critical harvest decisions for such highvalue products as wine grapes. As glucose is the most expensive carbonsource and feedstock in fermentation processes, glucose monitoring foroptimum reactor feed rate control is important in power alcoholproduction. Reactor mixing and control of glucose concentration also iscritical to quality control during production of soft drinks andfermented beverages, which consumes the largest amounts of glucose andfermentable (vicinal diol) sugars internationally.

[0070] When the indicator compounds incorporate fluorescent indicatorsubstituents, various detection techniques also are known in the art.For example, the compounds of the invention can be used in fluorescentsensing devices (e.g., U.S. Pat. No. 5,517,313) or can be bound topolymeric material such as test paper for visual inspection. This lattertechnique would permit, for example, glucose measurement in a manneranalogous to determining pH with a strip of litmus paper. The compoundsdescribed herein may also be utilized as simple reagents with standardbenchtop analytical instrumentation such as spectrofluorometers orclinical analyzers as made by Shimadzu, Hitachi, Jasco, Beckman andothers. These molecules would also provide analyte specificchemical/optical signal transduction for fiber optic-based sensors andanalytical fluorometers as made by Ocean Optics (Dunedin, Fla.), orOriel Optics.

[0071] U.S. Pat. No. 5,517,313, the disclosure of which is incorporatedherein by reference, describes a fluorescence sensing device in whichthe compounds of the present invention can be used to determine thepresence or concentration of glucose in a liquid medium. The sensingdevice comprises a layered array of a fluorescent indicatormolecule-containing matrix (hereafter “fluorescent matrix”), a high-passfilter and a photodetector. In this device, a light source, preferably alight-emitting diode (“LED”), is located at least partially within theindicator material, or in a waveguide upon which the indicator matrix isdisposed, such that incident light from the light source causes theindicator molecules to fluoresce. The high-pass filter allows emittedlight to reach the photodetector, while filtering out scattered incidentlight from the light source. The fluorescence of the indicator moleculesemployed in the device described in U.S. Pat. No. 5,517,313 ismodulated, e.g., attenuated or enhanced, by the local presence ofglucose.

[0072] In the sensor described in U.S. Pat. No. 5,517,313, the materialwhich contains the indicator molecule is permeable to the analyte. Thus,the analyte can diffuse into the material from the surrounding testmedium, thereby affecting the fluorescence emitted by the indicatorcompounds. The light source, indicator compound-containing material,high-pass filter and photodetector are configured such that at least aportion of the fluorescence emitted by the indicator compounds impactsthe photodetector, generating an electrical signal which is indicativeof the concentration of glucose in the surrounding medium.

[0073] In accordance with other possible embodiments for using theindicator compounds of the present invention, sensing devices also aredescribed in U.S. Pat. Nos. 5,910,661, 5,917,605 and 5,894,351, allincorporated herein by reference.

[0074] The compounds of the present invention can also be used in animplantable device, for example to continuously monitor blood glucoselevels in vivo. Suitable devices are described in, for example,co-pending U.S. patent application Ser. No. 09/383,148 filed Aug. 26,1999, as well as U.S. Pat. Nos. 5,833,603, 6,002,954 and 6,011,984, allincorporated herein by reference.

[0075] The compounds of the present invention can be prepared by personsskilled in the art without an undue amount of experimentation usingreadily known reaction mechanisms and reagents, for example includingreaction mechanisms which are consistent with the general proceduresdescribed below.

EXAMPLE 1

[0076] Water Soluble Copolymer of Anthracene Derivative and MAPTAC

[0077] I. Synthesis of Mono-Boronate-Anthracene Indicator co-Polymerizedin Water-Soluble Polymer:

[0078] A. 9-[3-(methacrylamido)propylamino]methylanthracene

[0079] To a suspension of N-(3-aminopropyl)methacrylamide hydrochloridesalt (11.82 g, 66.0 mmole, 3.0 equiv.) and DBMP (10 mg as inhibitor) in250 mL CHCl₃ at 0° C. was added dropwise DIEA (18.5 g, 25.0 mL, 144mmole, 6.5 equiv.) over a 20 min period. The mixture was allowed to warmto 25° C. and then recooled to 0° C. To the cooled mixture was addeddropwise a solution of 9-chloromethylanthracene (5.0 g, 22 mmole) inCHCl₃ (100 mL) over a 1 hour period. The mixture was subsequentlystirred at 25° C. for 1 hour, 50° C. for 12 hours and then 70° C. for 2hours. At this time, the mixture was washed with 4×60 mL portions ofwater, and the combined aqueous layers were extracted with CH₂Cl₂. Thecombined organic extracts were dried over anhydrous Na₂SO₄, decanted andconcentrated in vacuo. The crude material was purified by silica gelchromatography (flash silica gel, 2-5% CH₃OH/CH₂Cl₂) to yield 2.44 g(33%) of a solid product.

[0080] TLC: Merck silica gel 60 plates, Rf 0.39 with 90/10 CH₂Cl₂/CH₃OH,see with UV (254/366), ninhydrin stain.

[0081] B.9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3—(methacrylamido)propylamino]methylanthracene.

[0082] To a solution of9-[3-(methacrylamido)propylamino]-methylanthracene (2.44 g, 7.34 mmole)and DBMP (10 mg as inhibitor) in 200 mL CHCl₃ at 0° C. was added DIEA(2.85 g, 3.84 mL, 22.0 mmole, 3.0 equiv.) in portions over a 10 minperiod, followed by the dropwise addition of a solution of(2-bromomethylphenyl)boronic acid neopentyl ester (2.49 g, 8.81 mmole,1.2 equiv.) over a 30 min period. The mixture was subsequently stirredat 25° C. for 20 hours. At this time, the mixture was washed with water,and the combined aqueous layers were extracted with CH₂Cl₂. The combinedorganic extracts were dried over anhydrous Na₂SO₄, decanted andconcentrated in vacuo. The crude material was purified by silica gelchromatography (flash silica gel, 2-5% CH₃OH/CH₂Cl₂) to yield 2.50 g(76%) of a lightly yellow crystalline solid.

[0083] Mp: 72-73° C.

[0084] TLC: Merck silica gel 60 plates, Rf 0.36 with 90/10 CH₂Cl₂/CH₃OH,see with UV (254/366), ninhydrin stain.

[0085] C. Water Soluble Copolymer of9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-methylanthraceneand MAPTAC (1:20 Molar Ratio).

[0086] To a solution of9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methylanthracene(0.0490 g, 0.105 mmole) and [3-(methacrylamido)propyl]-trimethylammoniumchloride (MAPTAC, 50 wt % aqueous solution, 0.48 g, 0.90 mL, 2.1 mmole,20 equiv.) in 1.5 mL ethylene glycol was added 4,4′-azobis(cyanovalericacid) (0.008 g, 0.03 mmole, 1.4 mole % of total monomer). The solutionwas purged with argon gas for 5 minutes and then heated to 60° C. in thedark for 18 hours. At this time, the viscous solution was cooled to 25°C., diluted with 5 mL water and dialyzed through a cellulose acetatemembrane (MWCO 3500) against 3×4 L of water. The dialyzed material wasconcentrated to dryness to yield 0.339 g (68%) of a yellow glassy solid.

[0087] II. Modulation of Fluorescence with Glucose and Lactate

[0088] The modulation of the fluorescence of the copolymer (whichcontains a single recognition element) prepared in this example byglucose and lactate was determined. FIG. 1 shows the normalizedfluorescence emission (I/Io@420 nm) of 0.5 mg/mL solutions of thecopolymer (1:20 molar ratio) in PBS containing a) 0-20 mM glucose; b)0-20 mM lactate. Spectra were recorded using a Shimadzu RF-5301spectrafluorometer with excitation@365 nm; excitation slits at 1.5 nm;emission slits at 5 nm; ambient temperature. Error bars are standarddeviation with duplicate values for each data point. The fluorescence ofthe copolymer was affected by the presence of glucose and lactate.

EXAMPLE 2

[0089] Modulation of Bis-Boronate-Indicator Covalently Attached toWater-Soluble Polymer by Glucose and Potential PhysiologicalInterferences.

[0090] I. Synthesis of Single-Methacrylate Monomer ofBis-Boronate-Anthracene Indicator

[0091] A. 9,10-bis[[2-(2-hydroxyethoxy)ethylamino]methyl]-anthracene.

[0092] To a solution of 2-(2-aminoethoxy)ethanol (31.4 g, 30.0 mL, 299mmole, 20.9 equiv.) in 40 mL CHCl₃ at 23° C. was added9,10-bis(chloromethyl)anthracene (3.94 g, 14.3 mmole). The solution wasstirred in the dark for 67 hours. At this time, added 100 mL CH₂Cl₂ andwashed with 1×50 mL and 2×100 mL portions of NaHCO₃ (saturated aqueoussolution). The organic extract was dried over anhydrous Na₂SO₄, filteredand concentrated to yield 4.67 g (79%) of a yellow powder. Product (˜85%pure by RP-HPLC) was carried on as is.

[0093] HPLC conditions: HP 1100 HPLC chromatograph, Vydac 201TP 10×250mm column, 0.100 mL injection, 2 mL/min, 370 nm detection, A=water (0.1%HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18min, 80-100% B over 2 min, 100%B 2 min, retention time 15.6 min.

[0094] B.9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene.

[0095] A solution of9,10-bis[[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene (4.02 g, 9.75mmole), DIEA (12.6 g, 17.0 mL, 97.5 mmole, 10.0 equiv.) and(2-bromomethylphenyl)boronic acid neopentyl ester (13.7 g, 48 mmole, 4.9equiv.) in 125 mL CHCl₃ at 23° C. was stirred in the dark for 46 hours.At this time, the reaction mixture was concentrated initially by rotaryevaporation, then using a vacuum pump to remove the DIEA. The residuewas purified by alumina column chromatography (150 g activated neutralalumina, 0-3% CH₃OH/CH₂Cl₂) to yield 5.67 g (70%) of a viscous oil whichsolidified upon standing. Product (˜85% pure by RP-HPLC) was carried onas is.

[0096] TLC: Merck basic alumina plates, Rf 0.33 with 95/5 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0097] HPLC conditions: HP 1100 HPLC chromatograph, Vydac 201TP 10×250mm column, 0.100 mL injection, 2 mL/min, 370 nm detection, A=water (0.1%HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18min, 80-100% B over 2 min, 100% B 2 min, retention time 18.8 min.

[0098] C.9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]-methyl]anthracene.(Single-methacrylate Monomer)

[0099] A solution of9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]-methyl]anthracene(0.298 g, 0.359 mmole), methacrylic acid (0.304 g, 0.300 mL, 3.53 mmole,9.84 equiv.), DCC (0.965 g, 4.68 mmole, 13.0 equiv.) andN,N-dimethylaminopyridine (0.020 g, 0.16 mmole, 0.46 equiv.) in 15 mLCH₂Cl₂ at 23° C. was stirred in the dark for 4 hours. At this time, thereaction mixture was filtered and concentrated by rotary evaporation.The residue was purified by alumina column chromatography (50 gactivated neutral alumina, 0-4% CH₃OH/CH₂Cl₂) to yield 0.150 g (47%) ofa yellow solid.

[0100] FAB MS: Calc=d for C₅₂H₆₆B₂N₂0₉ [M]⁺ 885; Found [M+1]⁺ 886.

[0101] TLC: Merck basic alumina plates, Rf 0.45 with 95/5 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0102] HPLC: HP 1100 HPLC chromatograph,Vydac 201TP 10×250 mm column,0.100 mL injection, 2 mL/min, 370 nm detection, A=water (0.1% HFBA) andB=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18 min, 80-100%B over 2 min, 100% B 2 min, retention time 21 min.

[0103] D. Water Soluble Copolymer of 9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]-methyl]anthraceneand TMAMA (1:50 Molar Ratio).

[0104] To a solution of [2-(methacryloxy)ethyl]trimethylammoniumchloride (TMAMA, 70 wt % aqueous solution, 0.344 g monomer, 1.66 mmole,50 equiv.) in 0.600 mL water was added a solution of9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene(0.0024 g, 0.0033 mmole) in 3.00 mL MeOH. To this mixture was added4,4′-azobis(4-cyanovaleric acid) (0.0075 g, 0.027 mmole, 1.6 mole % oftotal monomer). The solution was filtered through a 0.45μ membranefilter, was purged with nitrogen gas and then heated in the dark at 55°C. for 16 hours. At this time, the viscous solution was cooled to 25° C.and concentrated in vacuo. The residue was diluted with 20 mL water andfiltered through a 0.2μ membrane filter. The polymer solution wasdialyzed through a cellulose acetate membrane (MWCO 3500) against 2×4 Lof water. From the dialysis was obtained 38.5 mL of polymer solution.Concentration of a portion of this solution to dryness indicated 0.0075g polymer per 1.0 mL solution. Overall 0.289 g (77%) yield of polymer.

[0105] II. Modulation of Fluorescence With Glucose, Lactate andAcetoacetate

[0106] The modulation of the fluorescence of the copolymer (whichcontains two recognition elements) prepared in this example by glucose,lactate and acetoacetate was determined. FIG. 2 shows the normalizedfluorescence emission (I/Io@428 nm) of a 1.5 mg/mL solution ofanthracene bis boronate-TMAMA (1:50 mole ratio) copolymer in PBScontaining a) 0-20 mM glucose; b) 0-20 mM lactate; c) 0-20 mM lithiumacetoacetate. Spectra were recorded using a Shimadzu RF-5301spectrafluorometer with excitation@365 nm; excitation slits at 1.5 nm;emission slits at 1.5 nm; ambient temperature. The fluorescence of thecopolymer was affected by the presence of glucose, but not by thepresence of lactate or acetoacetate.

EXAMPLE 3

[0107] Effect of Lactate in Solution on the Dose Response Effect ofGlucose on the Fluorescence of Bis-Boronate-Anthracene Indicator

[0108] A.9,10-bis[[2-(tert-butoxycarbonyl)ethylamino]methyl]-anthracene.

[0109] A solution of β-alanine tert-butyl ester hydrochloride (3.06 g,16.8 mmole, 5.09 equiv.), DIEA (4.27 g, 5.75 mL, 33.0 mmole, 10.00equiv.) and 9,10-bis(chloromethyl)anthracene (0.910 g, 3.31 mmole) in 75mL CHCl₃ at 23° C. was stirred in the dark for 93 hours. At this time,the solution was filtered and washed with 1×40 mL and 2×60 mL portionsof NaHCO₃ (saturated aqueous solution). The organic extract was driedover anhydrous Na₂SO₄, filtered and concentrated to yield a crude yellowsolid. The residue was purified by silica gel column chromatography (30g gravity grade gel, 0-3% CH₃OH/CH₂Cl₂) to yield 1.06 g (65%) of aviscous yellow-orange. Product was carried on as is.

[0110] TLC: Merck silica gel 60 plates, Rf 0.33 with 95/5 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0111] B.9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(tert-butoxycarbonyl)ethylamino]methyl]anthracene.

[0112] A solution of9,10-bis[[2-(tert-butoxycarbonyl)ethylamino]methyl]anthracene (1.60 g,3.25 mmole), DIEA (4.45 g, 6.00 mL, 34.4 mmole, 10.6 equiv.) and(2-bromomethylphenyl)boronic acid neopentyl ester (4.80 g, 17.0 mmole,5.22 equiv.) in 30 mL CHCl₃ at 23° C. was stirred in the dark for 4.5days. At this time, 45 mL CHCl₃ were added to the mixture and themixture was washed with 2×25 mL portions of NaHCO₃ (saturated aqueoussolution). The organic extract was dried over anhydrous Na₂SO₄, filteredand concentrated to yield crude reddish oil. The residue was purified byalumina column chromatography (100 g activated neutral alumina, 0-3%CH₃OH/CH₂Cl₂) to yield 3.5 g of an orange solid. The product wasdissolved, followed by the formation of a white precipitate (DIEA-HBrsalt). The solution was filtered and the filtrate concentrated to yield2.72 g (93%) of an orange solid. Product (>80% pure by RP-HPLC) wascarried on as is.

[0113] TLC: Merck basic alumina plates, Rf 0.66 with 95/5 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0114] HPLC conditions: HP 1100 HPLC chromatograph, Vydac 201TP 10×250mm column, 0.100 mL injection, 2 mL/min, 370 nm detection, A water (0.1%HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18min, 80-100% B over 2 min, 100% B 2 min, retention time 23.9 min.

[0115] C.9,10-bis[N-(2-boronobenzyl)-N-[2-(carboxyethyl)amino]-methyl]anthracene.

[0116] A solution of9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(tert-butoxycarbonyl)ethylamino]methyl]-anthracene(0.556 g, 0.620 mmole) in 5 mL 20% TFA/CH₂Cl₂ at 23° C. was stirred inthe dark for 25 hours. At this time, the reaction mixture wasconcentrated under a stream of N₂ gas. The residue was triturated with3×10 mL portions of ether. The residual solid was dried in vacuo toyield 0.351 g (87%) of a fluffy yellow powder.

[0117] FAB MS: Glycerol matrix; Calc=d for C₄₂H₄₆B₂N₂0₁₀ (bis glyceroladduct) [M]⁺ 760; Found [M]⁺ 760.

[0118] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.025 mL injection, 0.75 mL/min, 1.5 mL injection loop, 360 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 16.7 min.

[0119] D. Modulation of Fluorescence With Glucose and Lactate

[0120] The modulation of the fluorescence of the indicator compound(which contains two recognition elements) prepared in this example byglucose and lactate was determined. FIG. 3 shows the fluorescence (at428 nm) of 75 μM solutions of bis carboxylate bis-boronate-anthraceneindicator in PBS containing a) 0-10 mM glucose, 0 mM lactate; b) 0-10 mMglucose, 2 mM lactate; c) 0-10 mM glucose, 5 mM lactate. Spectra wererecorded using a Shimadzu RF-5301 spectrafluorometer with excitation@365nm; excitation slits at 1.5 nm; emission slits at 1.5 nm; ambienttemperature. All points measured in triplicate, with ±1 SD error barsincluded. The presence of lactate did not substantially affect thefluorescence modulation of the indicator by glucose.

EXAMPLE 4

[0121] Selectivity of Bis-Boronate Glucose Indicator for Glucose vs.Lactate and Acetoacetate when Indicator Covalently Immobilized in theHydrogel

[0122] I. Preparation of Dual-Methacrylamide Monomer

[0123] A. 9,10-bis[3-(methacrylamido)propylamino]-methylanthracene.

[0124] A suspension of 9,10-bis(chloromethyl)anthracene (1.5 g, 5.45mmole), DIEA (28.17 g, 38.00 mL, 218 mmole, 40 equiv.),N-(3-aminopropyl)methacrylamide hydrochloride salt (9.76 g, 54.5 mmole,10.0 equiv.), and ˜5 mg of BHT in 200 mL CHCl₃ at 23° C. was stirred inthe dark for 4 days at 40° C. At this time, the temperature wasincreased to 45° C. and the mixture was stirred for 3 days longer. Atthis time, a precipitate had formed. The mixture was filtered, and thesolid product dissolved in the minimum amount of CH₂Cl₂. A yellowcrystalline solid, the bis hydrochloride salt of the desired product,formed overnight (3.15 g, quantitative).

[0125] TLC: Merck basic alumina plates, Rf 0.31 with 90/10 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0126] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.100 mL injection, 0.75 mL/min, 360 nm detection, A=water (0.1%HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18min, 80-100% B over 2 min, 100% B 2 min, retention time 15.0 min.

[0127] B.9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methylanthracene.(Dual-methacrylamide Monomer)

[0128] A solution of9,10-bis[3-(methacrylamido)propylamino]methylanthracene (0.0.650 g, 1.34mmole of the free amine), DIEA (0.612 g, 0.825 mL, 4.74 mmole, 3.55equiv.), (2-bromomethylphenyl)boronic acid neopentyl ester (1.34 g, 4.74mmole, 3.55 equiv.) and BHT (5 mg as inhibitor) in 20 mL CHCl₃ at 23° C.was stirred in the dark for 5 days. At this time, the reaction mixturewas concentrated in vacuo and the residue was purified by aluminachromatography (200 g activated neutral alumina, 0-2% CH₃OH/CH₂Cl₂) toyield 0.465 g (39%) of a very viscous yellow oil.

[0129] TLC: Merck basic alumina plates, Rf 0.59 with 90/10 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0130] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.050 mL injection, 0.75 mL/min, 360 nm detection, A=water (0.1%HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18min, 80-100% B over 2 min, 100% B 2 min, retention time 16.9 min.

[0131] C. Preparation of N,N-dimethylacrylamide Hydrogel with GlucoseIndicator:

[0132] A solution of N,N-dimethylacrylamide (40% wt.) andN,N=-methylenebisacrylamide (0.8% wt.) in ethylene glycol was prepared.9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methylanthracene(17.8 mg, 2×10⁻⁵ mole) and 40 pL of aqueous ammonium persulfate (5% wt)were combined with 1 mL of ethylene glycol monomer solution. Theresulting solution was placed in a glove box purged with nitrogen. Anaqueous solution of N,N,N═,N=-tetramethylethylenediamine (80 μL, 5% wt.)was added to the monomer formulation to accelerate polymerization. Theresulting formulation was poured in a mold constructed from microscopeslides and 100 micron stainless steel spacer. After being kept for 8hours in nitrogen atmosphere the mold was placed in phosphate bufferedsaline (PBS) (10 mM PBS, pH=7.4), the microscope slides were separated,and the hydrogel was removed. The hydrogel was washed with 100 mL of PBScontaining 1 mM lauryl sulfate sodium salt and 1 mM EDTA sodium salt for3 days, the solution being changed every day, followed by washing withDMF/PBS (10/90 by vol., 3×100 mL), and finally with PBS (pH=7.4, 3×100mL). The resulting hydrogel polymer was stored in PBS (10 mM PBS,pH=7.4) containing 0.2% wt. sodium azide and 1 mM EDTA sodium salt.

[0133] II. Modulation of Fluorescence with Glucose, Lactate andAcetoacetate

[0134] The modulation of the fluorescence of the indicator compound(which contains two recognition elements) prepared in this example byglucose, lactate and acetoacetate was determined. FIG. 4 shows thenormalized fluorescence emission (I/Io@427 nm) of a hydrogel containingthe glucose recognition molecule of this example in 10 mM PBS, pH 7.4containing 0.2% NaN₃ and 1 mM EDTA containing various amounts ofsodium-L-lactate, lithium acetoacetate or α-D-glucose. Data wererecorded using a Shimadzu RF-5301 spectrofluorometer with excitation@365nm (slit=3 nm) and emission at 427 nm (slit=3 nm) at low sensitivity at37° C. using a temperature controlled sample holder. The cuvettescontaining 3 mL of the desired solution were equilibrated at 37° C. for15 minutes before measurement. Each hydrogel sample was measured in fourindependent samples. Error bars are standard deviation withquadruplicate values for each data point. The hydrogels containing aglucose recognition molecule were prepared as previously described. Thehydrogels were mounted on glass slides and covered with polyester meshin PMMA cuvettes at 45□ to the incident light. Solutions of 1, 5, 10 and20 mM sodium L-lactate [Aldrich], 5, 10 and 20 mM lithium acetoacetate[Aldrich], and 1, 2, 4, 5, 10, and 20 mM α-D-glucose were prepared in 10mM PBS, pH 7.4 containing 0.2% NaN₃ and 1 mM EDTA. The fluorescence ofthe copolymer was affected by the presence of glucose, but not by thepresence of lactate or acetoacetate.

EXAMPLE 5

[0135] Glucose Selectivity vs. Lactate Using Bis-Boronate Recognitionand Proximity Quenching Signal Generation

[0136] A. N-(2,2-diethoxyethyl)-4-bromo-1,8-naphthalimide.

[0137] A suspension of 4-bromo-1,8-naphthalic anhydride (10.0 g, 36.1mmol) and aminoacetaldehyde diethyl acetal (4.81 g, 5.26 mL, 36.1 mmol,1 equiv.) in 45 mL EtOH was stirred at 45° C. for 3 days. At this time,the resulting suspension was filtered, washing with EtOH and the residuewas dried to yield 13.3 g (94%) of a light brown solid product.

[0138] TLC: Merck silica gel 60 plates plates, Rf 0.17 with 98/2CH₂Cl₂/CH₃OH, see with UV (254/366).

[0139] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.050 mL injection, 0.75 mL/min, 1.5 mL injection loop, 360 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 24.2 min.

[0140] B. N-(2,2-diethoxyethyl)-4-butylamino-1,8-naphthalimide.

[0141] A solution of N-(2,2-diethoxyethyl)-4-bromo-1,8-naphthalimide(0.797 g, 2.03 mmol) and n-butylamine (1.48 g, 2.00 mL, 20.2 mmol, 9.96equiv.) in 8 mL NMP was heated at 45° C. for 66 hours. At this time, theresulting suspension was allowed to cool to 25° C., followed byfiltration. The residue was dissolved with 50 mL ether and extracted3×50 mL water. The organic extract was dried over anhydrous Na₂SO₄,filtered and concentrated to yield a crude yellow powder. The crudematerial was purified by silica gel chromatography (25 g gravity gradegel, 0-1% CH₃OH/CH₂Cl₂) to yield 0.639 g (82%) of a yellow powder.

[0142] TLC: Merck silica gel 60 plates, Rf 0.71 with 95/5 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0143] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.050 mL injection, 0.75 mL/min, 1.5 mL injection loop, 450 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 23.5 min.

[0144] C. N-(2-oxoethyl)-4-butylamino-1,8-naphthalimide.

[0145] A solution ofN-(2,2-diethoxyethyl)-4-butylamino-1,8-naphthalimide (0.622 g, 1.62mmol) and p-toluene-sulfonic acid mono hydrate (0.010 g, 0.053 mmol,0.032 equiv.) in 25 mL acetone was stirred at 25° C. for 18 hours. Atthis time, the solution was concentrated and the residue purified bysilica gel chromatography (25 g gravity grade gel, 0-1% CH₃OH/CH₂Cl₂) toyield 0.470 g (94%) of an orange solid.

[0146] TLC: Merck silica gel 60 plates, Rf 0.61 with 95/5 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0147]¹H NMR (400 MHZ, CDCl₃); δ 1.03 (t, 3H, J=7.3 Hz), 1.53 (m, 2H),1.78 (m, 2H), 3.38 (t, 2H, J=7.2 Hz), 5.02 (s, 2H), 6.64 (d, 1H, J=8.6Hz), 7.52 (dd, 1H, J=7.4, 8.3 Hz), 8.08 (dd, 1H, J=1 Hz, 8.5 Hz), 8.38(d, 1H, J=8.3 Hz), 8.46 (dd, 1H, J=1.0, 7.3 Hz), 9.75 (s, 1H).

[0148] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.050 mL injection, 0.75 mL/min, 1.5 mL injection loop, 450 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 19.6 min.

[0149] D. N-(4-dimethylaminobenzyl)-1,6-diaminohexane.

[0150] A suspension of 4-dimethylaminobenzaldehyde (1.00 g, 6.70 mmol),Na₂SO₄ (6.70 g, 47.2 mmol, 7.04 equiv.) and 1,6-diaminohexane (3.89 g,33.5 mmol, 5.00 equiv.) in 20 mL anhydrous EtOH was stirred in the darkat 25° C. under an atmosphere of nitrogen gas for 18 hours. At thistime, the solution was filtered and NaBH₄ (1.73 g, 45.8 mmol, 6.84equiv.) was added to the filtrate. The suspension was stirred at 25° C.for 5 hours. At this time, the reaction mixture was concentrated and theresidue dissolved in 50 mL water and extracted 3×50 mL ether. Thecombined organic extracts were washed 2×50 mL water. The combinedaqueous extracts were extracted 2×50 mL ether. The combined organicextracts were dried over Na₂SO₄, filtered and concentrated to yield 1.35g (81%) of a viscous oil.

[0151] TLC: Merck silica gel 60 plates plates, Rf 0.58 with 80/15/5CH₂Cl₂/CH₃OH/iPrNH₂, see with ninhydrin stain, UV (254/366).

[0152] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.050 mL injection, 0.75 mL/min, 1.5 mL injection loop, 280 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 13.3 min.

[0153] E. N-2-[6-N(N-4-dimethylaminobenzyl)aminohexyl]aminoethyl)-4-butylamino-1,8-naphthalimide.

[0154] To a suspension of N-(2-oxoethyl)-4-butylamino-1,8-naphthalimide(0.346 g, 1.11 mmol) in 25 mL anhydrous MeOH was added a solution ofN-(4-dimethylaminobenzyl) -1,6-diaminohexane (0.554 g, 2.22 mmol, 2.00equiv.) and acetic acid (0.067 g, 1.1 mmol, 1.0 equiv.) in 20 mLanhydrous MeOH. To this mixture was added a solution of NaCNBH₃ (0.070g, 1.1 mmol, 1.0 equiv.) in 5 mL anhydrous MeOH. The reaction mixturewas stirred at 25° C. for 15 hours. At this time, the MeOH was removedby rotary evaporation and the residue was dissolved in 30 mL water. Thesolution was adjusted to pH 2 with 1 N HCl and then stirred for 1 hourat 25° C. At this time, the solution was adjusted to pH 12 with 1 N NaOHand subsequently extracted 3×50 mL CH₂Cl₂. The combined organic extractswere washed 3×50 mL water, dried over anhydrous Na₂SO₄, filtered andconcentrated to yield a crude brown oil. The crude material was purifiedby silica gel chromatography (35 g flash grade gel, 0-50% CH₃OH/CH₂Cl₂,then 45/50/5 CH₃OH/CH₂Cl₂/iPrNH₂) to yield 0.190 g (32%) of diamineproduct.

[0155] FAB MS: Calc=d for C₃₃H₄₅N₅O₂ [M]⁺ 544; Found [M]⁺ 544.

[0156] TLC: Merck silica gel 60 plates, Rf 0.42 with 80/20 CH₂Cl₂/CH₃OH,see with ninhydrin stain and UV (254/366).

[0157] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.050 mL injection, 0.75 mL/min, 1.5 mL injection loop, 450 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 17.6 min.

[0158] F.N-2-[6-N-(N-4-dimethylaminobenzyl)-6-N-[2-(5,5-dimethylborinan-2-yl)benzyl]aminohexyl]-[2-(5,5-dimethylborinan-2-yl)benzyl]aminoethyl-4-butylamino-1,8-naphthalimide.

[0159] To a solution ofN-2-[6-N-(N-4-dimethylaminobenzyl)aminohexyl]aminoethyl)-4-butylamino-1,8-naphthalimide(0.150 g, 0.276 mmole) and DIEA (0.355 g, 0.478 mL, 2.81 mmole, 10.0equiv.) in 5 mL CHCl₃ was added a solution of(2-bromomethylphenyl)boronic acid neopentyl ester (0.390 g, 1.38 mmole,5.00 equiv.) in 2 mL CHCl₃. The solution was subsequently stirred at 25°C. for 27 hours. At this time, the mixture was concentrated and theresidue was purified by alumina column chromatography (100 g activatedneutral alumina, 0-5% CH₃OH/CH₂Cl₂) to yield 0.024 g (19%) of a viscousbrown oil.

[0160] FAB MS (glycerol matrix): Calc'd for C₅₃H₆₇B₂N₅O₈ [M]⁺ 924 (bisglycerol adduct in place of bis neopentyl ester of boronic acids); Found[M]⁺ 924.

[0161] TLC: Merck neutral alumina plates, Rf 0.62 with 80/20CH₂Cl₂/CH₃OH, see with UV (254/366).

[0162] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.050 mL injection, 0.75 mL/min, 1.5 mL injection loop, 450 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 20.7 min.

[0163] G.N-2-[6-N-(N-4-dimethylaminobenzyl)-6-N-[2-(borono)benzyl]aminohexyl]-[2-(borono)benzyl]aminoethyl-4-butylamino-1,8-naphthalimide (nBuF-hexa-Q bis-boronate).

[0164] The free bis boronic acid product used in glucose studies resultsfrom dissolution ofN-2-[6-N-(N-4-dimethyl-aminobenzyl)-6-N-[2-(5,5-dimethylborinan-2-yl)benzyl]amino-hexyl]-[2-(5,5-dimethylborinan-2-yl)benzyl]aminoethyl-4-butylamino-1,8-naphthalimidein the MeOH/PBS buffer system.

[0165] H. Modulation of Fluorescence with Glucose and Lactate.

[0166] The modulation of the fluorescence of the indicator compound(which contains two recognition elements) prepared in this sample byglucose and lactate was determined. FIG. 5 shows the normalizedfluorescence emission (I/Io@535 nm) of 0.015 mM solutions of theindicator compund in 70/30 MeOH/PBS containing a) 0-20 mM glucose; b)0-20 mM lactate. Spectra were recorded using a Shimadzu RF-5301spectrafluorometer with excitation@450 nm; excitation slits at 1.5 nm;emission slits at 1.5 nm; ambient temperature. Error bars are standarddeviation with triplicate values for each data point. The fluorescenceof the indicator was affected by the presence of glucose, but notsubstantially affected by the presence of lactate.

EXAMPLE 6

[0167] Effect of Glucose or Lactate on Acrylamide Gel ContainingN-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamide(Alizarin Red S Monomer) andα,α′-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene(bis Boronic Acid Monomer):

[0168] A. 3,4-Dihydroxy-9,10-dioxo-2-anthracenesulfonyl Chloride:

[0169] 3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonic acid sodium salt(1.4 g, 3.9 mmoles) was combined with 30 mL of chlorosulfonic acid andheated to 90° C. for 5 hours, after which the solution was cooled to 0°C. and poured into 100 g of ice. After the ice melted the solution wasextracted with CH₂Cl₂ (3×100 mL), methylene chloride extracts werecombined, dried with Na₂SO₄ and evaporated to produce 0.87 g of solid(Yield 66%).

[0170] B.N-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamide:

[0171] 3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonyl chloride (96 mg,0.28 mmoles) and N-(3-aminopropyl) methacrylamide hydrochloride (108 mg,0.6 mmoles) were combined with 20 mL of CH₂Cl₂. To this suspension Et₃N(303 mg, 3 mmoles) was added. The mixture was stirred at roomtemperature for 24 hours, filtered, and solvent was evaporated. Theresulting solid was subjected to column chomatography on SiO₂ (10 g)with CH₂Cl₂/MeOH (90/10) as an eluent. The product was obtained as a redsolid (80 mg, 64% yield).

[0172] FAB MS: Calculated for C₂₁H₂₀N₂O₇S M⁺ 445; Found M⁺ 445.

[0173] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.100 mL injection, 0.75 mL/min, 2 mL injection loop, 370 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 17.67 min.

[0174] C. α,α′-bis[3-(methacrylamido)propylamino]-1,4-xylene.

[0175] A solution of N-(3-aminopropyl)methacrylamide hydrochloride salt(3.00 g, 16.8 mmole, 2.21 equiv.), DIEA (6.5 g, 8.8 mL, 50 mmole, 6.6equiv.), terephthaldicarboxaldehyde (1.02 g, 7.60 mmole) and Na₂SO₄(10.7 g, 75.3 mmole, 9.91 equiv.) in 75 mL anhydrous MeOH was stirred inthe dark at 25□C for 18 hours. At this time, more Na₂SO₄ (10.7 g, 75.3mmole, 9.91 equiv.) was added and stirring continued for 6 hours longer.At this time, the solution was filtered and NaBH₄ (1.73 g, 45.7 mmole,6.01 equiv.) was added to the filtrate in portions and subsequentlystirred at 25° C. for 21 hours. The suspension was filtered throughCelite and the filtrate was concentrated. The residue was dissolved in100 mL CH₂Cl₂ and washed 1×25 mL saturated aqueous NaHCO₃. The organicextract was dried over anhydrous Na₂SO₄, filtered and concentrated toyield a viscous oil. The product was carried on as is.

[0176] HPLC: HP 1100 HPLC chromatograph, Vydac 201TP 10×250 mm column,0.100 mL injection, 2.00 mL/min, 260 nm detection, A=water (0.1% HFBA)and B=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18 min,80-100% B over 2 min, 100% B 2 min, retention time 15.8 min.

[0177] D.α,α=-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene.

[0178] A solution of α,α=-bis[3-(methacrylamido)propylamino]-1,4-xylene(2.94 g, 7.61 mmole), DIEA (2.97 g, 4.00 mL, 23.0 mmoles, 3.02 equiv.),(2-bromomethylphenyl)boronic acid neopentyl ester (6.50 g, 23.0 mmole,3.02 equiv.) and BHT (5 mg as inhibitor) in 75 mL CH₂Cl₂ at 25° C. wasstirred in the dark for 28 hours. At this time, the mixture was washed1×25 mL saturated aqueous NaHCO₃. The organic extract was dried overanhydrous Na₂SO₄, filtered and concentrated. To the residue was added200 mL ether and the suspension was stirred for 18 hours. The suspensionwas filtered and the residue dissolved in CH₂Cl₂, filtered and thefiltrate concentrated. To the solid residue was added 150 mL ether andthe suspension was stirred for 18 hours. At this time, the suspensionwas filtered yielding 1.98 g (33%) of a fluffy pink powder.

[0179] FAB MS: Calc=d for C₄₆H₆₄B₂N₄0₆ [M]⁺ 790; Found [M+1]⁺ 791.

[0180] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.050 mL injection, 0.75 mL/min, 280 nm detection, A=water (0.1%HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18min, 80-100% B over 2 min, 100% B 2 min, retention time 13.4 m/n.

[0181] E. Preparation of Acrylamide Gel ContainingN-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamide(Alizarin Red S Monomer) andα,α′-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene:

[0182] Ethylene glycol solution containing 30% wt. acrylamide and 0.8%wt. N,N′-methylenebisacrylamide was prepared.

[0183] N-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamide (1.5 mg, 3.38×10⁻⁶ mole) andα,α′-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene(28 mg, 3.54×10⁻⁵ mole) were combined with 800 μL of ethylene glycolmonomer solution and 40 μL of 5% wt. aqueous ammonium persulfate. Thisformulation was placed in a glove box purged with nitrogen along with amold constructed from glass microscope slides and 100 micron stainlesssteel spacer. An aqueous solution ofN,N,N′,N′-tetramethylethylenediamine (40 μL, 5% wt.) was added to themonomer solution to accelerate polymerization and the final formulationwas poured into a glass mold. The mold was left under nitrogenatmosphere for 16 hours, after which it was immersed in PBS (pH=7.4) andthe glass slides were separated to afford a hydrogel polymer in a formof a thin film. The resulting hydrogel thin film was washed with 100 mLof phosphate buffered saline containing 1 mM lauryl sulfate sodium saltfor 3 days, the solution being changed every day, followed by washingwith MeOH/PBS (20/80 by vol., 3×100 mL), and finally with PBS (pH=7.4,3×100 mL). Hydrogel polymer was stored in PBS (10 mM PBS, pH=7.4)containing 0.2% wt. sodium azide and 1 mM EDTA sodium salt.

[0184] F. Modulation of Absorbance with Glucose and Lactate

[0185] The modulation of the absorbance of the indicator hydrogel (whichcontains two recognition elements) prepared in this example by glucoseand lactate was determined. The acrylamide gel was mounted in PMMA cellin the same way as described in Example 4. Phosphate buffered saline(PBS), pH=7.4 containing desired amount of glucose or sodium lactate washeated to 37° C. in a water bath and placed in the PMMA cell containingthe gel after which the PMMA cell was allowed to equilibrate for 15 minat 37° C. Absorbance measurement for each glucose or lactateconcentration was conducted in triplicate. For each measurement,absorbance at 650 nm was used as a blank, A(650 nm) was subtracted fromall values of A(450 nm) and A(530 nm).

[0186]FIG. 6 shows the absorbance spectra for acrylamide gel (30%)containing 4 mM Alizarin Red S monomer and 44 mM bis boronic acidmonomer with and without glucose. FIG. 7 shows the effect of glucose onabsorbance of acrylamide gel (30%) containing 4 mM Alizarin Red Smonomer and 44 mM bis boronic acid monomer. FIG. 8 shows the effect ofsodium lactate on absorbance of acrylamide gel (30%) containing 4 mMAlizarin Red S monomer and 44 mM bis boronic acid monomer. Theabsorbance of the indicator was affected by the presence of glucose, butnot substantially affected by the presence of lactate.

[0187] G. Modulation of Fluorescence with Glucose and Lactate

[0188] The modulation of the fluorescence of an acrylamide gelsynthesized substantially in accordance with this Example 6 (except that1.9 mg ofN-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamideand 35 mg ofα,α′-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylenewere used) was determined.

[0189] The experiment was conducted in a Shimadzu RF-5301 PCspectrofluorimeter equipped with a variable temperature attachment(excitation at 470 nm, slits {fraction (3/10)} nm, high sensitivity).The acrylamide gel was attached to a piece of a glass slide which wasglued in a PMMA fluorescence cell at a 45° angle. The cell was filledwith 2.5 ml of PBS (pH=7.4) and heated to 37° C. Stock solutions ofglucose (100 mM and 500 mM) in PBS (pH=7.4) were prepared and heated to37° C. in a water bath. An aliquot of heated glucose stock solution wasadded to the PMMA cell periodically while the fluorescence intensity at550 nm was monitored as a function of time (1 measurement every 2minutes). Glucose concentration in the PMMA cell was measured using aYSI Model 2300 STAT plus glucose analyzer. The results, shown in FIG. 9,show that the addition of glucose reduces the fluorescent intensity ofthe indicator hydrogel. The same effect is seen in FIG. 10, which showsthe effect of glucose on the fluorescence spectrum of the same type ofgel.

[0190] That effect is believed to occur because of the followingconsiderations. The methacrylamide monomer of Alizarin Red S (reportermolecule) contains a vicinal diol functionality and monomerfunctionality (see structure below). In aqueous solution and in organicsolvents, the Alizarin Red S and bis-boronate recognition elementmonomers (see structure below) are capable of reversible reaction witheach other to form a boronate ester. The boronate ester molecule formedin this reversible reaction is fluorescent, while the Alizarin Red Smonomer by itself displays virtually no fluorescence emission in aqueoussolution and in organic solvents, such as MeOH. Thus upon binding to theglucose recognition element, Alizarin Red S changes its opticalproperties, such as absorbance and quantum yield of fluorescence, forexample.

[0191] A solution of Alizarin Red S with monomer functionality andglucose recognition element with monomer functionality can be preparedtogether with a hydrogel monomer and a crosslinker. Copolymerization ofthis mixture produces a hydrogel material which is diffusable to varioussmall and medium size molecules; thus it is capable of analyte detectionand quantitation. An analyte, such as glucose for example, would diffuseinside the hydrogel matrix and displace the reporter molecule previouslybound to the recognition element. This event causes a change in theoptical properties of the hydrogel film since it now contains a greaternumber of reporter molecules unbound to the recognition element.

[0192] The modulation of the fluorescence of the indicator compound(which contains two recognition elements) prepared in this example byglucose and lactate was also determined. The experiment was conducted ina Shimadzu RF-5301 PC spectrofluorimeter equipped with a variabletemperature attachment (excitation at 470 nm, slits {fraction (5/10)}nm, low sensitivity). The acrylamide gel was attached to a piece of aglass slide which was glued in a PMMA fluorescence cell at a 45° angle.The cell was filled with 2.5 ml of PBS (pH=7.4) and heated to 37° C. ina water bath. A stock solution of sodium lactate (100 mM) in PBS(pH=7.4) was prepared and heated to 37° C. in a water bath. Stocksolutions of glucose (100 mM and 500 mM) in PBS (pH=7.4) were preparedand heated to 37° C. in a water bath. An aliquot of heated lactate stocksolution was added to the PMMA cell periodically while the fluorescenceintensity at 550 nm was monitored as a function of time (1 measurementevery 2 minutes), until the lactate concentration reached 8 mM. Then, analiquot of heated glucose stock solution was added to the PMMA cellperiodically while the fluorescence intensity at 550 nm was monitored asa function of time (1 measurement every 2 minutes). Glucoseconcentration in the PMMA cell was measured using a YSI Model 2300 STATplus glucose analyzer. The results, shown in FIG. 11, show that theaddition of lactate had no significant effect on the fluorescentintensity of the indicator hydrogel, and the subsequent addition ofglucose reduced the fluorescent intensity of the indicator hydrogel.

EXAMPLE 7

[0193] Single-Methacrylamide Monomer of Bis-Boronate-Anthracene:

[0194] A.9-chloromethyl-10-[[2-(2-hydroxyethoxy)ethylamino]-methyl]anthraceneHydrochloride Salt.

[0195] To a suspension of 9,10-bis(chloromethyl)anthracene (5.18 g, 18.8mmole, 3.99 equiv.) in 200 mL of NMP was added 2-(2-aminoethoxy)ethanol(0.495 g, 0.475 mL, 4.71 mmole). The mixture was stirred in the dark for17 hours. At this time, the reaction mixture was concentrated to ˜50 mLunder vacuum at 50° C. The residue was purified by silica gelchromatography (150 g gravity grade silica gel, 0-10% CH₃OH/CH₂Cl₂) toyield 0.425 g (24%) of a yellow/orange solid. TLC: Merck silica gel 60plates, Rf 0.72 with 70/30 CH₂Cl₂/CH₃OH, see with UV (254/366),ninhydrin stain. HPLC: HP 1100 HPLC chromatograph, Vydac 201TP 10×250 mmcolumn, 0.100 mL injection, 2 mL/min, 370 nm detection, A=water (0.1%HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18min, 80-100% B over 2 min, 100% B 2 min, retention time 16.1 min.

[0196] B.9-[[2-(2-hydroxyethoxy)ethylamino]methyl]-10-[[(3-methacrylamido)propylamino]methyl]-anthracene.

[0197] To a suspension of N-(3-aminopropyl)methacrylamide hydrochloridesalt (3.08 g, 17.2 mmole, 4.2 equiv.), DIEA (5.19 g, 7.00 mL, 40.1mmole, 9.8 equiv.) and ˜3 mg of BHT in 125 mL CHCl₃ at 23° C. was addeddropwise a solution of9-chloromethyl-10-[[2-(2-hydroxyethoxy)ethylamino]-methyl]anthracenehydrochloride salt (1.56 g, 4.10 mmole) in 25 mL of CHCl₃. The mixturewas subsequently stirred in the dark for 92 hours. At this time, thereaction mixture was filtered and washed with 2×40 mL of NaHCO₃(saturated aqueous solution). The organic extract was dried overanhydrous Na₂SO₄, filtered and concentrated to yield a sticky orangesolid which was purified by alumina chromatography (50 g activatedneutral alumina, 0-5% CH₃OH/CH₂Cl₂) to yield 0.364 g (20%) of an orangesolid.

[0198] TLC: Merck silica gel 60 plates, Rf 0.16 with 70/30 CH₂Cl₂/CH₃OH,see with UV (254/366), ninhydrin stain

[0199] HPLC: HP 1100 HPLC chromatograph, Vydac 201TP 10×250 mm column,0.100 mL injection, 2 mL/min, 370 nm detection, A=water (0.1% HFBA) andB=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18 min, 80-100%B over 2 min, 100% B 2 min, retention time 16.85 min.

[0200] C.9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene.(Single-Methacrylamide Monomer)

[0201] A solution of9-[[2-(2-hydroxyethoxy)ethylamino]-methyl]-10-[[(3-methacrylamido)propylamino]methyl]-anthracene(0.343 g, 0.763 mmole), DIEA (0.965 g, 1.30 mL, 9.8 equiv.) and(2-bromomethylphenyl)boronic acid neopentyl ester (1.09 g, 3.85 mmole,5.0 equiv.) in 20 mL CHCl₃ at 23° C. was stirred in the dark for 25hours. At this time, the reaction mixture was concentrated initially byrotary evaporation, then using a vacuum pump to remove DIEA. The residuewas purified by alumina column chromatography (40 g activated neutralalumina, 0-10% CH₃OH/CH₂Cl₂) to yield 0.299 g (46%) of a yellow orangesolid. This compound may be co-polymerized with a suitable monomer asdescribed previously, deprotected, and used to detect glucose.

[0202] FAB MS: Calc=d for C₅₁H₆₅B₂N₃0₇ [M]⁺ 854; Found [M+1]⁺ 855.

[0203] TLC: Merck basic alumina plates, Rf 0.35 with 95/5 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0204] HPLC: HP 1100 HPLC chromatograph, Vydac 201TP 10×250 mm column,0.100 mL injection, 2 mL/min, 370 nm detection, A=water (0.1% HFBA) andB=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18 min, 80-100%B over 2 min, 100% B 2 min, retention time 19.7 min.

EXAMPLE 8

[0205] Dual-Methacrylate Monomer of Bis-Boronate-Anthracene

[0206] A.9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino] methyl]anthracene.

[0207] A solution of9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]-anthracene(0.100 g, 0.120 mmole; see Example 2), methacrylic acid (0.112 g, 0.110mL, 1.30 mmole, 10.8 equiv.), DCC (0.316 g, 1.53 mmole, 12.8 equiv.) andN,N-dimethylamino-pyridine (0.014 g, 0.11 mmole, 0.92 equiv.) in 5 mLCH₂Cl₂ was stirred at 0° C. for 1 hour, then 23° C. for 22 hours. Atthis time, the reaction mixture was filtered and concentrated by rotaryevaporation. The residue was purified by alumina column chromatography(30 g activated neutral alumina, 0-2% CH₃OH/CH₂Cl₂) to yield 0.030 g(26%) of a yellow solid. This compound may be co-polymerized with asuitable monomer as described previously, deprotected, and used todetect glucose.

[0208] FAB MS: Calc=d for C₅₆H₇₀B₂N₂0₁₀ [M]⁺ 953; Found [M]⁺ 951 (weakmolecular ion peak).

[0209] TLC: Merck basic alumina plates, Rf 0.67 with 95/5 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0210] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.100 mL injection, 0.75 mL/min, 2 mL injection loop, 370 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 19.6 min.

EXAMPLE 9

[0211] Dual 5-Aminopentyl Bis-Boronate-Anthracene

[0212] A. 9,10-bis[[5-(t-BOC)-aminopentylamino]methyl]-anthracene.

[0213] A suspension of 9,10-bis(chloromethyl)anthracene (0.28 g, 1mmole), DIEA (7.0 mL, 40 mmole), mono-t-butoxycarbonyl1,5-diaminopentane (3.75 g, 10 mmole), and 50 ml of CHCl₃ was stirred inthe dark for 2 days at 45° C. The solution was washed with saturatedH₂O/NaHCO₃, the organic phase was dried (Na₂SO₄), and the solvent wasevaporated. The residue was purified by alumina chromatography (40 gactivated neutral alumina, 95/5% vol. CH₂Cl₂/MeOH) to yield 0.55 g ofviscous oil. This material was used as is for the next step.

[0214] B.9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[5-(t-BOC)-aminopentylamino]methyl]anthracene.

[0215] A solution of9,10-bis[[5-(t-BOC)-aminopentylamino]-methyl]anthracene (0.3 g, 0.49mmole), DIEA (0.35 mL, 2 mmole), and (2-bromomethylphenyl)boronic acidneopentyl ester (0.566 g, 2.0 mmole) in 20 mL CH₂Cl₂ was stirred in thedark for 2 days at 25° C. At this time, the reaction mixture wasconcentrated in vacuo and the residue was purified by aluminachromatography (60 g of activated neutral alumina, 98/2% vol.CH₂Cl₂/MeOH) to yield 0.401 g of yellow oil. This material was used asis for the next step.

[0216] C.9,10-bis[N-(2-boronobenzyl)-N-[5-aminopentylamino]-methyl]anthraceneTrifluoroacetic Acid Salt.

[0217]9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[5-(t-BOC)-aminopentylamino]methyl]anthracene(0.4 g, 0.39 mmole) was dissolved in 20 ml of CH₂Cl₂/TFA (80/20% vol.).The solution was stirred for 12 hours, the solvent was evaporated, andthe residue was washed with 10 ml of ether. A total of 373 mg of solidwas obtained (72% yield). Product was 80% pure by RP-HPLC. This compoundmay be co-polymerized with a suitable monomer as described previously,deprotected, and used to detect glucose.

[0218] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.050 mL injection, 0.75 mL/min, 360 nm detection, A=water (0.1%HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18min, 80-100% B over 2 min, 100% B 2 min, retention time 16.0 min.

EXAMPLE 10

[0219]

[0220] A.N-2-(tert-butoxycarbonyl)aminoethyl-4-bromonaphthalene-1,8-dicarboximide:

[0221] N-t-Boc-ethylenediamine (Fluka, 1.6 g, 10 mmole) and4-bromo-1,8-naphthalic anhydride (Aldrich, 2.77 g, 10 mmole) werecombined with 60 ml of anhydrous ethanol, the suspension was stirred at60° C. for 20 hours, cooled to room temperature, and filtered. Theobtained solid was washed with 30 ml of cold EtOH and dried undervacuum. Yield 3.84 g (91%). NMR (CDCl₃): 1.28 (9H, s); 3.52 (2H, t);4.35 (2H, t); 4.92 (1H, s);7.84 (1H, t); 8.04 (1H, d); 8.42 (1H, d);8.58 (1H, d); 8.67 (1H, d)

[0222] B.N-2-(tert-butoxycarbonyl)aminoethyl-4-(N′-methylaminoethylamino)naphthalene-1,8-dicarboximide:

[0223] N-Methylethylenediamine (1.48 g, 20 mmole) was combined with 2 mlof 1-methyl-2-pyrrolidinone (NMP) followed by addition ofN-2-(tert-butoxycarbonyl)aminoethyl-4-bromonaphthalene-1,8-dicarboximide(0.35 g, 0.845 mmole). The resulting solution was stirred at 45° C. for40 hours after which NMP and N-methylethylenediamine were evaporatedunder vacuum. The obtained residue was subjected to columnchromatography (20 g of silica gel, initially CH₂Cl₂/MeOH (90/10), thenCH₂Cl₂/MeOH/Et₃N (75/20/5)). A yellow solid was obtained (0.311 g, 89%yield). Purity was checked by RP-HPLC.

[0224] C.N-aminoethyl-4-(N′-aminoethylene-N″-[2-(borono)benzyl]methylamino)naphthalene-1,8-dicarboximideTrifluoroacetic Acid Salt:

[0225]N-2-(tert-butoxycarbonyl)aminoethyl-4-(N′-methylaminoethylamino)naphthalene-1,8-dicarboximide(0.3 g, 0.73 mmole), 2-bromomethylphenyl boronic acid, pinacol ester(0.6 g, 2 mmole), N,N-diisopropyl-N-ethylamine (1.3 ml, 8 mmole), and 10ml of CH₂Cl₂ were combined. The solution was stirred for 20 hours,followed by addition of 2 g of PS-Trisamine resin (ArgonautTechnologies, 3.38 mmol/g). The reaction mixture and resin were agitatedfor 10 hours after which the resin was removed by filtration and washedwith CH₂Cl₂ (2×20 ml). Combined CH₂Cl₂ solutions were evaporated anddried under vacuum.

[0226] Methylene chloride solution containing 20% vol. TFA and 5% vol.triisopropyl silane was added to the resulting orange residue. Theresulting solution was stirred at room temperature for 10 hours, afterwhich the solvent was evaporated and the residue triturated with etherto yield a yellow solid. The solid was filtered and dried in vacuum(yield 580 mg). Purity of the material was checked by RP-HPLC. The solidwas used as is in the next step.

[0227] D.N-(3-Borono-5-nitrobenzamido)ethyl-4-(N′-aminoethylene-N″-[2-(borono)benzyl]methylamino)naphthalene-1,8-dicarboximide:

[0228]N-aminoethyl-4-(N′-aminoethylene-N″-[2-(borono)benzyl]methylamino)naphthalene-1,8-dicarboximidetrifluoroacetic acid salt (0.225 g, 0.4 mmole),3-carboxy-5-nitrophenylboronic acid (0.085 g, 0.4 mmole),diphenylphosphoryl azide (0.13 ml, 0.6 mmole), and 2 ml of anhydrous DMFwere combined. N,N-diisopropyl-N-ethyl amine (0.7 ml, 4 mmole) was addedand the solution was stirred for 20 hours. Ether (10 ml) was added tothe reaction mixture and the insoluble residue was separated andsonicated with 5 ml of CH₂Cl₂ to yield an orange solid which wasfiltered and dried under vacuum (38 mg, 15% yield). Purity of the solidwas checked by RP-HPLC. NMR (dmso-d6/D2O, 90/10): δ 2.32 (3H, s); 2.82(2H, t); 3.58 (2H, t); 3.65 (2H, t), 3.70 (2H, s); 6.65 (1H, d); 7.0-7.3(4H, m); 7.68 (1H, t); 8.18 (1H, d); 8.42 (1H, d); 8.47 (1H, d);8.1-8.35 (3H, m).

[0229] E. Test ofN-(3-borono-5-nitrobenzamido)ethyl-4-(N′-aminoethylene-N″-[2-(borono)benzyl]methylamino)naphthalene-1,8-dicarboximidefor Interaction with Glucose as Monitored by Fluorescence

[0230] This experiment was conducted in MeOH/phosphate buffered saline,(PBS, 10 mM, pH=7.4). The concentration ofN-(3-borono-5-nitrobenzamido)ethyl-4-(N′-aminoethylene-N″-[2-(borono)benzyl]methylamino)naphthalene-1,8-dicarboximidein MeOH/PBS, (50/50 vol. %) was 15 M. The glucose concentration wasvaried from 0 mM to 50 mM, and the L-sodium lactate concentration wasvaried from 0 mM to 7 mM. The experiment was conducted in a ShimadzuRF-5301 PC spectrofluorimeter: excitation wavelength was set at 430 nm,emission was monitored in the 480-650 nm range, slit width 3/1.5 nm,high sensitivity of PMT.

[0231] The results are shown in FIGS. 12 and 13, which show that thefluorescence of the indicator of this example was affected by thepresence of glucose, but not by the presence of lactate.

EXAMPLE 11

[0232] 6-(Cyclohexanecarboxamido)hexylamine Indicator Monomer

[0233] A.9-[N-[3-(methacrylamido)propylamino]methyl]-10-N-[(6-aminohexylamino)methyl]anthracene.To a solution of 3-aminopropylmethacrylamide (0.775 g, 5.45 mmol, 10.0equiv.) and tert-butyl N-(6-aminohexyl)carbamate (1.18 g, 5.45 mmol,10.0 equiv.) and several crystals of BHT in 200 mL CHCl₃ was added9,10-bis(chloromethyl)anthracene (0.150 g, 0.545 mmol). The reactionmixture was subsequently stirred in the dark at ambient temperature for4 days. At this time, the CHCl₃ was evaporated and the residue wasdissolved in 100 mL ether. The organic layer was extracted with 8×125 mLsat'd aqueous NaHCO₃ and 5×200 mL phosphate buffer (0.4 M, pH 7.0). ThepH of the combined phosphate buffer washes was adjusted to pH 11 byaddition of Na₂CO₃ (sat'd aqueous solution), followed by extraction with5×300 mL CH₂Cl₂. The combined organic layers were concentrated and theresidue dissolved in 5 mL of a 20% solution of TFA in CH₂Cl₂. Themixture was stirred at ambient temperature for 2 hours. At this time,the reaction mixture was extracted with 4×10 mL sat'd aqueous NaHCO₃.The pH of the combined aqueous layers was adjusted to pH 11 by additionof Na₂CO₃ (sat'd aqueous solution), followed by extraction with 4×75 mLCH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄,filtered and concentrated in vacuo to yield 0.068 g (27%) of product.

[0234] TLC: a) Merck Silica Gel 60 plates, Rf 0.16 with 70/30CH₂Cl₂/CH₃OH, see with UV (254/366), prior to deprotection; Rf 0.27 with85/14.5/0.5 CH₂Cl₂/CH₃OH/iPrNH₂, see with UV (254/366), final product.

[0235] HPLC: HP 1100 HPLC chromatograph, Waters 8×100 mm NovaPak HR C18column, 0.100 mL injection, 0.75 mL/min, 0.400 mL injection loop, 360 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 15.5 min.

[0236] B.9-[N-[3-(methacrylamido)propylamino]methyl]-10-[N-[6-(cyclohexanecarboxamido)hexylamino]methyl]anthracene.

[0237] To a solution of9-[N-[3-(methacrylamido)propylamino]methyl]-10-N-[6-aminohexylamino)methyl]anthracene(1.68 g, 3.63 mmol) and a few crystals of BHT in 20 mL CH₂Cl₂ at ambienttemperature was added dropwise a solution of cyclohexanecarboxylic acidN-hydroxysuccinimide ester (0.845 g, 3.76 mmol, 1.03 equiv.) over a 1hour period. The reaction was subsequently stirred in the dark atambient temperature for 16 hours. At this time, the reaction mixture wasconcentrated in vacuo and the residue dissolved in 105 mL of a solutionof 90/15 ether/CH₂Cl₂. The organic layer was extracted with 4×225 mLphosphate buffer (0.4 M, pH 7.0). The pH of the combined phosphatebuffer washes was adjusted to pH 11 by addition of Na₂CO₃ (sat'd aqueoussolution), followed by extraction with 6×500 mL CH₂Cl₂. The combinedorganic layers were dried over anhydrous Na₂SO₄, filtered andconcentrated in vacuo to yield 1.2 g (60%) of product.

[0238] TLC: Merck Silica Gel 60 plates, Rf 0.30 with 85/14.5/0.5CH₂Cl₂/CH₃OH/iPrNH₂, see with UV (254/366)

[0239] HPLC: HP 1100 HPLC chromatograph, Waters 8×100 mm NovaPak HR C18column, 0.100 mL injection, 0.75 mL/min, 0.400 mL injection loop, 360 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 17.4 min.

[0240] C.9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[6-(cyclohexanecarboxamido)hexylamino]-methyl]anthracene.A solution of9-[N-[3-(methacrylamido)propylamino]methyl]-10-[N-[6-(cyclohexanecarboxamido)hexylamino]methyl]-anthracene(1.0 g, 1.8 mmol), DIEA (1.81 g, 2.44 mL, 14.0 mmol, 7.8 equiv.),2-bromomethylphenylboronic acid pinacol ester (2.14 g, 7.20 mmol, 4.0equiv.) and a few crystals of BHT in 30 mL CHCl₃ was stirred in the darkat ambient temperature for 60 hours. At this time, the reaction mixturewas concentrated and the residue(9-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[6-(cyclohexanecarboxamido)hexylamino]methyl]anthracene)suspended in 150 mL ether. The organic layer was washed with 4×50 mLphosphate buffer (0.4 M, pH 7.0). The organic layer was concentrated andthe residue dissolved in ether in 200 mL 0.1 N aqueous HCl. The aqueouslayer was washed with 3×50 mL 1:1; ether:ethyl acetate and the pH wasadjusted to pH 11 by addition of Na₂CO₃ (sat'd aqueous solution),followed by extraction with 3×150 mL CH₂Cl₂. The combined organic layerswere dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo toyield a red oily compound. The residue was dissolved in ether andconcentrated in vacuo to yield 1.17 g (85%) of a yellow solid product.

[0241] TLC: Merck Silica Gel 60 plates, Rf 0.59 with 80/20 CH₂Cl₂/CH₃OH,see with UV (254/366)

[0242] HPLC: HP 1100 HPLC chromatograph, Waters 8×100 mm NovaPak HR C18column, 0.100 mL injection, 0.75 mL/min, 0.400 mL injection loop, 360 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 19.6 min.

[0243]¹H NMR (9:1 d₆-acetone/D₂O): δ 0.90 (m, 2H), 1.03 (m, 2H),1.18-1.30 (m, 6H), 1.35-1.48 (4H), 1.62 (m, 1H, O═C—CH(CH₂)CH₂),1.66-1.75 (m, 7H), 1.77 (m, 2H, N—CH₂—CH ₂—CH₂—N), 2.52 (m, 2H, N—CH₂—CH₂—), 2.63 (m, 2H, N—CH ₂—CH₂—), 2.98 (m, 4H, —CH ₂—NH—C═O), 3.98 (s,4H, benzene-CH ₂—N), 4.57 (s, 2H, athracene-CH ₂—N), 4.59 (s, 2H,athracene-CH ₂—N), 5.20 (t, 1H, J=1.5 Hz, C═CH ₂), 5.46 (s, 1H, C═CH ₂),7.4-7.5 (m, 8H, Ar—H), 7.52 (m, 2H, Ar—H), 7.95 (m, 2H, Ar—H), 8.23 (m,4H, Ar—H)

[0244] D. N,N-dimethylacrylamide Hydrogel with9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[6-(cyclohexanecarboxamido)hexylamino]methyl]anthracene. A solution of N,N-dimethylacrylamide (40% wt.) andN,N′-methylenebisacrylamide (0.8% wt.) in phosphate buffer, pH=7.4, 200mM was prepared.9-[N-(2-Boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[6-(cyclohexanecarboxamido)hexylamino]methyl]anthracene (18 mg, 2.15×10⁻⁵ mole) and 60 mg of fructose werecombined with 2 mL of MeOH. This solution was sonicated until all of thefructose dissolved and was subsequently evaporated to yield a solid. Tothis solid, 1 mL of phosphate buffer solution containing monomers wasadded. After sonication for 10 min this solution was filtered through a0.2 μM PTFE membrane filter. Aqueous ammonium persulfate (20 μL, 5% wt.)was combined with the formulation. The resulting solution was placed inglove box purged with nitrogen. An aqueous solution ofN,N,N′,N′-tetramethylethylenediamine (40 μL, 5% wt.) was added to themonomer formulation to accelerate polymerization. The resultingformulation was poured into a mold constructed from glass microscopeslides and a 100 μM stainless steel spacer. After being kept for 8 hoursin a nitrogen atmosphere, the mold was placed in phosphate bufferedsaline (pH=7.4), the microscope slides were separated, and the hydrogelwas removed. The hydrogel was washed with 100 mL of phosphate bufferedsaline (PBS) containing 1 mM lauryl sulfate sodium salt and 1 mM EDTAtetrasodium salt for 3 days, the solution being changed every day,followed by washing with EtOH/PBS (20/80 by vol., 3×100 mL), and finallywith PBS (H=7.4, 3×100 mL). The resulting hydrogel film was stored inPBS (pH=7.4) containing 0.02% wt. sodium azide and 1 mM EDTA tetrasodiumsalt.

[0245] E. Modulation of Fluorescence with Glucose.

[0246] The modulation of the fluorescence of the6-(cyclohexanecarboxamido)hexylamine indicator/DMA hydrogel filmprepared in this example by glucose and lactate was determined. FIG. 14shows the relative fluorescence emission (I@430 nm) of the hydrogel filmin PBS (pH 7.4 containing 0.02% NaN₃ and 1 mM EDTA) containing 0 to 20mM α-D-glucose, 0 to 10 mM L-sodium lactate, and 0-20 mM α-D-glucose inthe presence of 4 mM L-sodium lactate. The hydrogel film (100 μmthickness, 8 mm diameter disk) was mounted in a PMMA cuvette at a 45°angle. All measurements were made at 37° C. in a Shimadzu RF-5301spectrofluorometer with excitation at 370 nm (slit=3 nm) and emission at430 nm (slit=3 nm) at low PMT sensitivity. Glucose and L-sodium lactateconcentrations were checked using the YSI Model 2300 STAT plus glucoseanalyzer. Error bars are standard deviation with triplicate values foreach data point. The fluorescence was affected by the presence ofglucose, but not by the presence of lactate. Moreover, the presence oflactate (4 mM) had no significant effect on the 0-20 mM glucosecalibration curve.

EXAMPLE 12 2-(Carboxyethyl)Amine Indicator Monomer

[0247]

[0248] Chemical Name:9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(carboxyethyl)amino]methyl]anthracene(uncapped)

[0249] Chemical Formula: C₄₀H₄₅B₂N₃O₇

[0250] MW: 701.4

[0251] Physical appearance: faint yellow powder

[0252] Solubility: PBS/methanol, methanol, ethanol, chloroform,dichloromethane

[0253] Capped Indicator:9-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[2-(carboxyethyl)amino]methyl]anthracene.

[0254] I. Synthesis

[0255] A.9-[N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(tert-butoxycarbonyl)ethylamino]methyl]anthracene.To a solution of 3-aminopropylmethacrylamide (12.9 g, 90.7 mmol, 4.99equiv.), β-alanine tert-butyl ester (13.2 g, 90.9 mmol, 5.00 equiv.) andseveral crystals of BHT in 700 mL CHCl₃ was added9,10-bis(chloromethyl)anthracene (5.00 g, 18.2 mmol). The reactionmixture was subsequently stirred in the dark at 30° C. for 88 hours. Atthis time, the CHCl₃ was evaporated and the residue was dissolved in 500mL ether. The solution was stirred for 1 hour at which time salts hadprecipitated from solution. The ether solution was filtered andsubsequently extracted with 10×350 mL sat'd aqueous NaHCO₃. The etherlayer was further extracted with 6×350 mL phosphate buffer (0.2 M, pH6.5). The pH of the combined phosphate buffer washes was adjusted to pH11-12 by addition of Na₂CO₃ (sat'd aqueous solution), followed byextraction with 6×500 mL CH₂Cl₂. The combined organic layers were driedover anhydrous Na₂SO₄, filtered and concentrated in vacuo to yield anoily crude product. The crude product was purified by silica gelchromatography (50 g flash grade silica gel, 0-5% MeOH/CH₂Cl₂ stepgradient) to yield 2.04 g of a sticky yellow solid (23%).

[0256] TLC: Merck Silica Gel 60 plates, Rf 0.29 with 90/10 CH₂Cl₂/CH₃OH,see with UV (254/366) and ninhydrin stain.

[0257] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.100 mL injection, 0.75 mL/min, 1.500 mL injection loop, 280 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 17.0 min.

[0258] B.9-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[2-(tert-butoxycarbonyl)ethylamino]methyl]anthracene.

[0259] A solution of9-[N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(tert-butoxycarbonyl)ethylamino]methyl]anthracene(1.5 g, 3.1 mmol), DIEA (3.16 g, 4.26 mL, 24.4 mmol, 7.9 equiv.),2-bromomethylphenylboronic acid pinacol ester (3.64 g, 12.2 mmol, 3.9equiv.) and a few crystals of BHT in 50 mL CHCl₃ was stirred in the darkat ambient temperature for 16 hours. At this time, the reaction mixturewas concentrated and the residue suspended in 200 mL ether. The etherlayer was extracted with 3×125 mL phosphate buffer (0.2 M, pH 7.0),dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo to yielda crude product. The residue was triturated with hexanes to yield 2.14 g(76%) of a white solid.

[0260] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.200 mL injection, 0.75 mL/min, 1.500 mL injection loop, 280 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 19.2 min.

[0261] C.9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(carboxyethyl)amino]methyl]anthracene.

[0262] A solution of9-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[2-(tert-butoxycarbonyl)ethylamino]methyl]anthracene(0.294 g, 0.319 mmol) in 5 mL of 20% TFA/CH₂Cl₂ was stirred in the darkat ambient temperature for 22 hours. At this time, the reaction mixturewas concentrated and the residue triturated with ether. The residue wasdissolved in 5 mL 90:10 acetone/water and stirred for 2 hours. At thistime, the reaction mixture was concentrated and the residue trituratedwith water and PBS (pH 7.4 containing 0.02% NaN₃ and 1 mM EDTA)resulting in the recovery of 0.062 g (28%) of a light yellow solid.

[0263] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.100 mL injection, 0.75 mL/min, 1.500 mL injection loop, 280 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 17.4 min.

[0264] FAB MS: Glycerol matrix; Calc'd for C₄₆H₅₃B₂N₃O₇ (bis glyceroladduct) [M]⁺ 813; Found [M+2]^(+ 815.)

[0265] D. N,N-dimethylacrylamide hydrogel with9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(carboxyethyl)amino]methyl]anthracene.A solution of N,N-dimethylacrylamide (40% wt.) andN,N′-methylenebisacrylamide (0.8% wt.) in phosphate buffer, pH=7.4, 200mM was prepared.9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(carboxyethyl)amino]methyl]anthracene(14 mg, 2.0×10⁻⁵ mole) and 60 mg of fructose were combined with 2 ml ofMeOH. This solution was sonicated until all fructose dissolved andevaporated to yield a solid. To this solid 1 ml of phosphate buffersolution containing monomers was added. After sonication for 10 minutesthis solution was filtered through 0.2 μM PTFE filter. Aqueous ammoniumpersulfate (20 1L, 5% wt.) was combined with the formulation. Theresulting solution was placed in a glove box purged with nitrogen. Anaqueous solution of N,N,N′,N′-tetrametylethylenediamine (40 μL, 5% wt.)was added to the monomer formulation to accelerate polymerization. Theresulted formulation was poured in a mold constructed from microscopeslides and 100 [M stainless steel spacer. After being kept for 8 hoursin nitrogen atmosphere the mold was placed in phosphate buffered saline(10 mM, pH=7.4), the microscope slides were separated, and the hydrogelwas removed. The hydrogel was washed with 100 ml of phosphate bufferedsaline (PBS) containing 1 mM lauryl sulfate sodium salt and 1 mM EDTAtetrasodium salt for 3 days, the solution being changed every day,followed by washing with EtOH/PBS (20/80 by vol., 3×100 ml), and finallywith PBS (pH=7.4, 3×100 ml). The resulting hydrogel film was stored inPBS (10 mM, pH=7.4) containing 0.02% wt. sodium azide and 1 mM EDTAtetrasodium salt.

[0266] II. Modulation of Fluorescence with Glucose.

[0267] The modulation of the fluorescence of the 2-(carboxyethyl)amineindicator/DMA hydrogel film prepared in this example by glucose andlactate was determined. FIG. 15 shows the relative fluorescence emission(I@430 nm) of the hydrogel film in PBS (pH 7.4 containing 0.02% NaN₃ and1 mM EDTA) containing 0 to 20 mM α-D-glucose, 0 to 10 mM L-sodiumlactate, and 0-20 mM glucose in the presence of 3 mM L-sodium lactate.The hydrogel film (100 μm thickness, 8 mm diameter disk) was mounted ina PMMA cuvette at a 45° angle. All measurements were made at 37° C. in aShimadzu RF-5301 spectrofluorometer with excitation at 370 nm (slit=3nm) and emission at 430 nm (slit=3 nm) at low PMT sensitivity. Glucoseand L-sodium lactate concentrations were checked using the YSI Model2300 STAT plus glucose analyzer. The data is plotted as the average oftriplicate values for each data point. The fluorescence was affected bythe presence of glucose, but not by the presence of lactate. Moreover,the presence of lactate (4 mM) had no significant effect on the 0-20 mMglucose calibration curve.

EXAMPLE 13 Fluorescent Glucose Indicator Containing Two DetectableMoieties:

[0268]

[0269] Chemical Name:9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[3-(N-6-(9-anthracenecarboxamido)hexylaminocarbonyl)ethylamino]methyl]anthracene (uncapped)

[0270] Chemical Formula: C₇₃H₈₇B₂N₅O₇

[0271] MW: 1168

[0272] Physical appearance: faint yellow powder

[0273] Solubility: PBS/methanol, methanol, ethanol, chloroform,dichloromethane

[0274] Pinacol Capped Compound:

[0275]9-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(N-6-(9-anthracenecarboxamido)hexylaminocarbonylethylaminomethyl]anthracene.

[0276] I. Synthesis

[0277] A. 9-Anthracenoyl Chloride: Anthracene-9-carboxylic acid (1.2 g,5.4×10⁻³ mole) was combined with 15 ml of thionyl chloride. The solutionwas refluxed for 2 hours followed by evaporation of the volatilecomponents. The obtained solid was dried under high vacuum for 24 hoursyielding 1.3 g of material (quantitative yield). This material was usedas is in the next step.

[0278] B. N-(6-Aminohexyl)-anthracene-9-carboxamide hydrochloric acidsalt: 9-Anthracenoyl chloride (1.3 g, 5.4 mmole) in 50 ml of anhydrousCH₂Cl₂ was added dropwise to 11.6 g of hexamethylenediamine (100 mmole)in 100 ml of CH₂Cl₂ at 0° C. The solution was stirred at 0° C. for 1hour then allowed to warm to room temperature and stirred overnight. Thesolvent was evaporated and 200 ml of water was added to the residue.This mixture was sonicated and stirred for 1 hour then filtered. Thefiltered solid was dried under vacuum for 24 hours. MeOH (50 ml) and 2ml of conc. HCl was added to the solid, followed by evaporation of MeOH.The resulting solid was washed with hot CH₂Cl₂/MeOH (90/10 vol. %) andrecrystallized from MeOH, yielding 0.51 g of the product (26%). Thepurity of the product was checked by HPLC.

[0279] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.1 mL injection, 0.75 mL/min flowrate, 2 mL injection loop, 280nm detection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B2 min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min,retention time 16.5 min.

[0280] C.9-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(N-6-(9-anthracenecarboxamido)hexylaminocarbonylethylaminomethyl]anthracene:

[0281] 9-[N-[2-(4,4,5,5, -tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[2-carboxyethylamino]methyl]anthracene(40 mg, 4.5×10⁻⁵ mole) was combined withN-(6-aminohexyl)-anthracene-9-carboxamide hydrochloric acid salt (20 mg,5.6×10⁻⁵ mole), diphenylphosphoryl azide (15.4 mg, 5.6×10⁻⁵ mole), and 2ml of DMF. Diisopropylethylamine(39 20 μL, 2.44×10⁻⁴ mole) was added tothe mixture and the solution was stirred at room temperature for 24hours. The DMF was evaporated under high vacuum, the residue wasdissolved in 50 ml of EtOAc and washed with water (3×10 ml). The EtOAcsolution was separated, dried (Na₂SO₄), and evaporated producing 46 mgof solid (87% yield). Purity of the material was checked by HPLC.

[0282] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.1 mL injection, 0.75 mL/min flowrate, 2 mL injection loop, 280nm detection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B2 min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min,retention time 20.42 min.

[0283] FAB mass-spectrum, glycerol matrix: calculated for C₆₇H₇₅B₂N₅O₉(bis glycerol adduct) [M]+=1116, found [M+1]+=1117.

[0284] II. Effect of Glucose on Fluorescence of Indicator Immobilized inHydrogel Film

[0285] Preparation of HEMA/Methacrylic Acid Hydrogel with GlucoseIndicator:

[0286] A 50% wt. solution of 2-hydroxyethylmethacrylate(4.75 g) andmethacrylic acid (0.25 g) in phosphate buffer, pH=7.4, 200 mM wasprepared. Glucose indicator (11 mg, 1.0×10⁻⁵ mole) and 60 mg of fructosewere combined with 2 ml of MeOH. This solution was sonicated until allfructose dissolved and evaporated to yield a solid. To this solid 1 mlof phosphate buffer solution containing monomers was added. Aftersonication for 10 minutes this solution was filtered through 0.2 μM PTFEfilter. Aqueous ammonium persulfate (20 μL, 5% wt.) was combined withthe formulation. The resulting solution was placed in a glove box purgedwith nitrogen. An aqueous solution ofN,N,N′,N′-tetramethylethylenediamine (40 μL, 5% wt.) was added to themonomer formulation to accelerate polymerization. The resultingformulation was poured in a mold constructed from microscope slides anda 100 μM stainless steel spacer. After being kept for 8 hours in anitrogen atmosphere the mold was placed in phosphate buffered saline (10mM, pH=7.4), the microscope slides were separated, and the hydrogel wasremoved. The hydrogel was washed with 100 ml of phosphate bufferedsaline (PBS) containing 1 mM lauryl sulfate sodium salt and 1 mM EDTAtetrasodium salt for 3 days, the solution being changed every day,followed by washing with EtOH/PBS (20/80 by vol., 3×100 ml), and finallywith PBS (pH=7.4, 3×100 ml). The resulting hydrogel film was stored inPBS (10 mM, pH=7.4) containing 0.02% wt. sodium azide and 1 mM EDTAtetrasodium salt.

[0287] Effect of Glucose and L-Sodium Lactate on Hydrogel FilmContaining Glucose Indicator.

[0288] The experiment was conducted in a Shimadzu RF-5301 PCspectrofluorimeter equipped with a variable temperature attachment.Excitation wavelength was set at 370 nm, slits 3/3 nm, low PMTsensitivity, emission was scanned from 400 to 600 nm. Glucose andL-sodium lactate concentrations were checked using YSI Model 2300 STATplus glucose analyzer.

[0289] The hydrogel film (100 μm thickness, round shape—8 mm diameter)was mounted in a PMMA cuvette at 45° angle. Phosphate buffered saline(PBS), pH=7.4 containing the desired amount of glucose, L-sodiumlactate, and glucose with L-sodium lactate were heated to 37° C. in awater bath and placed in the PMMA cell containing the mounted hydrogel.After each addition the PMMA cell was allowed to equilibrate for 45 minat 37° C. Fluorescence intensity measurements for each glucose/lactateconcentration were conducted on two different samples and an averagevalue was used in the calibration curve. Calibration curves(Fluorescence Intensity at 430 nm vs. concentration) were obtained forglucose, L-sodium lactate, and glucose in the presence of 3 mM L-sodiumlactate. The results are shown in FIG. 16.

EXAMPLE 14 6-(3-Carboxypropionamido)Hexylamino Indicator Monomer

[0290]

[0291] Pinacol Capped Compound:

[0292]9-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[6-(3-carboxypropionamido)hexylamino]methyl]anthracene.

[0293] Uncapped Compound:

[0294]9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[6-(3-carboxypropionamido)hexylamino]methyl]anthracene.

[0295] Synthesis:

[0296] The synthesis may be carried out in an analogous fashion toExample 11 using9-[N-[3-(methacrylamido)propylamino]methyl]-10-N-[6-(hexylamino)methyl]anthraceneas the starting material. In contrast, the amine starting material isreacted with the N-hydroxysuccinimide (NHS) ester of the mono methylester of succinic acid in place of the NHS ester ofcyclohexanecarboxylic acid used in Example 11. An additional basehydrolysis step is required to complete the synthesis.

EXAMPLE 15 Glucose Indicator/Monomer Excited with Visible Light

[0297]

[0298] Chemical Name:N-(3-Methacrylamidopropyl)-4-[2-N-[[2-(borono)benzyl]-[6-(N-[2-(borono)benzyl]-6-N-(3-carboxypropanamidoethyl)aminohexyl]]aminoethylamino]naphthalene-1,8-dicarboximide

[0299] Chemical Formula: C₄₇H₆₀B₂N₆O₁₀

[0300] M. W.: 890

[0301] The compound may be synthesized as shown below:

EXAMPLE 16 Alternate Glucose Indicator/Monomer Excited with VisibleLight

[0302]

[0303] Chemical Name:N-Butyl-4-[2-N-[[2-(borono)benzyl]-[6-(N-[2-(borono)benzyl]-6-N-(2-1methacrylamidoethyl)aminohexyl]]aminoethylamino]naphthalene-1,8-dicarboximide

[0304] Chemical Formula: C₄₄H₅₇B₂N₅O₇

[0305] M. W.: 789.5

[0306] The compound may be synthesized as shown below:

What is claimed is:
 1. A method for detecting the presence orconcentration of glucose in a sample which may also contain analpha-hydroxy acid or a beta-diketone, which comprises: a) exposing thesample to a compound having at least two recognition elements forglucose, oriented such that the interaction between the compound andglucose is more stable than the interaction between the compound and thealpha-hydroxy acid or beta-diketone, said compound also containing adetectable moiety having a detectable quality that changes in aconcentration-dependent manner when said compound is exposed to glucosein said sample; and b) measuring any change in said detectable qualityto thereby determine the presence or concentration of glucose in saidsample, wherein the presence of the alpha-hydroxy acid or thebeta-diketone does not substantially interfere with said determination.2. The method of claim 1, wherein the compound has the followingstructure:

wherein: R₁ and R₂ are the same or different and are selected from thefollowing: i) hydrogen; ii) a substituent to modify the pKa andhydrolytic stability of the R₈ moiety, iii) a detectable moiety, or iv)a linking group capable of attachment to a solid support or a polymericmatrix, said support or matrix optionally containing a detectablemoiety; R₃ is hydrogen or a linking group capable of attachment to asolid support or a polymeric matrix, said support or matrix optionallycontaining a detectable moiety; R₄ and R₅ are the same or different andare selected from the following: i) hydrogen, ii) a substituent tomodify the pKa and hydrolytic stability of the R₈ moiety, iii) adetectable moiety, or iv) a linking group capable of attachment to asolid support or a polymeric matrix, said support or matrix optionallycontaining a detectable moiety; each Z is independently carbon ornitrogen; R₆ and R₇ are the same or different and are i) linking groupshaving from zero to ten contiguous or branched carbon and/orheteroatoms, or ii) a linking group capable of attachment to a solidsupport or a polymeric matrix, said support or matrix optionallycontaining a detectable moiety; R is selected from the following: i) analiphatic and/or aromatic spacer containing from 1 to 10 contiguousatoms selected from the group consisting of carbon, oxygen, nitrogen,sulfur and phosphorus, ii) a detectable moiety, or iii) a linking groupcapable of attachment to a solid support or a polymeric matrix, saidsupport or matrix optionally containing a detectable moiety; each R₈ isthe same or different and is a moiety capable of interaction with thevicinal diol groups present in glucose; and R₉ and R₁₀ are the same ordifferent, and are i) hydrogen, ii) a detectable moiety, iii) a groupwhich is a) a linking group capable of attachment to a solid support ora polymeric matrix, said support or matrix optionally containing adetectable moiety, and/or b) includes a functional group capable ofaltering the physical properties of the compound; with the proviso thatthe indicator compound contains at least one detectable moietyassociated therewith.
 3. The method of claim 2, wherein R₈ is selectedfrom the group consisting of boronic acid, boronate ion, arsenious acid,arsenite ion, telluric acid, tellurate ion, germanic acid, germanateion, and combinations thereof.
 4. The method of claim 3, wherein each R₈is a boronic acid group.
 5. The method of claim 2, wherein the compoundcomprises at least two detectable moieties that are capable of energytransport from one to the other, and wherein said energy transport ismodulated by the presence of glucose in the sample.
 6. The method ofclaim 2, wherein at least one of R, R₁, R₂, R₄, R₅, R₉ or R₁₀ comprisesa fluorophore moiety and further wherein at least one of those groupscomprises a quenching moiety, and wherein said fluorophore is eitherquenched or dequenched when said compound interacts with glucose in thesample.
 7. The method of claim 2, wherein the compound comprises afluorophore, and the fluorescence of said fluorophore is modulated bythe interaction of said compound with glucose.
 8. The method of claim 1,wherein the sample is a physiological fluid.
 9. The method of claim 8,wherein the physiological fluid is selected from the group consisting ofblood, plasma, serum, interstitial fluid, cerebrospinal fluid, urine,saliva, intraocular fluid, lymph, tears, sweat, and physiologicalbuffers.
 10. The method of claim 1, wherein the compound is exposed tothe sample in solution.
 11. The method of claim 1, wherein the compoundis immobilized on or within a solid support.
 12. The method of claim 11,wherein the solid support is a polymeric matrix.
 13. The method of claim1, wherein the compound is associated with an implantable device, andwherein step a) takes place in vivo.
 14. The method of claim 2, whereinR is an anthracene residue; R₁, R₂, R₃, R₄ and R₅ are hydrogen; R₆ andR₇ are dimethylamine residues; each R₈ is a boronic acid group; one orboth of R₉ and R₁₀ are aliphatic carboxylic acid residues; and each Z iscarbon.
 15. The method of claim 14, wherein one or both of R₉ and R₁₀are propionic acid residues.
 16. The method of claim 2, wherein R is ahexamethylene residue; R₁, R₂, R₃, R₄ and R₅ are hydrogen; R₆ and R₇ aredimethylamine residues; each R₈ is a boronic acid group; R₉ is anaphthalimide residue; R₁₀ is a dimethylaminobenzyl residue; and each Zis carbon.
 17. The method of claim 2, wherein R is an anthraceneresidue; R₁, R₂, R₃, R₄ and R₅ are hydrogen; R₆ and R₇ are dimethylamineresidues; each R₈ is a boronic acid group; R₉ and R₁₀ are the same ordifferent and are selected from the group consisting of amethacrylamidoalkyl residue, a methacroyloxyethoxyalkyl residue, ahydroxyethoxyalkyl residue, and an aminoalkyl residue; and each Z iscarbon.
 18. The method of claim 2, wherein the compound is selected fromthe group consisting of:9-[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[2-(carboxyethyl)amino]-methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methylanthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[5-aminopentylamino]-methyl]anthracene;and 9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[6-(cyclohexanecarboxamido)hexylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(carboxyethyl)amino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[3-(N-6-(9-anthracenecarboxamido)hexylaminocarbonyl)ethylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[6-(3-carboxypropionamido)hexylamino]methyl]anthracene;N-(3-Methacrylamidopropyl)-4-[2-N-[[2-(borono)benzyl]-[6-(N-[2-(borono)benzyl]-6-N-(3-carboxypropanamidoethyl)aminohexyl]]aminoethylamino]naphthalene-1,8-dicarboximide;N-Butyl-4-[2-N-[[2-(borono)benzyl]-[6-(N-[2-(borono)benzyl]-6-N-(2-methacrylamidoethyl)aminohexyl]]aminoethylamino]naphthalene-1,8-dicarboximide; and salts thereof.
 19. A compound having thefollowing structure

wherein: R₁ and R₂ are the same or different and are selected from thefollowing: i) hydrogen; ii) a substituent to modify the pKa andhydrolytic stability of the R₈ moiety, iii) a detectable moiety, or iv)a linking group capable of attachment to a solid support or a polymericmatrix, said support or matrix optionally containing a detectablemoiety; R₃ is hydrogen or a linking group capable of attachment to asolid support or a polymeric matrix, said support or matrix optionallycontaining a detectable moiety; R₄ and R₅ are the same or different andare selected from the following: i) hydrogen, ii) a substituent tomodify the pKa and hydrolytic stability of the R₈ moiety, iii) adetectable moiety, or iv) a linking group capable of attachment to asolid support or a polymeric matrix, said support or matrix optionallycontaining a detectable moiety; each Z is independently carbon ornitrogen; R₆ and R₇ are the same or different and are i) linking groupshaving from zero to ten contiguous or branched carbon and/orheteroatoms, or ii) a linking group capable of attachment to a solidsupport or a polymeric matrix, said support or matrix optionallycontaining a detectable moiety; R is selected from the following: i) analiphatic and/or aromatic spacer containing from 1 to 10 contiguousatoms selected from the group consisting of carbon, oxygen, nitrogen,sulfur and phosphorus, ii) a detectable moiety, or iii) a linking groupcapable of attachment to a solid support or a polymeric matrix, saidsupport or matrix optionally containing a detectable moiety; each R₈ isthe same or different and is an optionally protected moiety which whenunprotected is capable of interaction with the vicinal diol groupspresent in glucose; and R₉ and R₁₀ are the same or different, and are i)hydrogen, ii) a detectable moiety, iii) a group which is a) a linkinggroup capable of attachment to a solid support or a polymeric matrix,said support or matrix optionally containing a detectable moiety, and/orb) includes a functional group capable of altering the physicalproperties of the compound; with the proviso that the indicator compoundcontains at least one detectable moiety associated therewith.
 20. Thecompound of claim 19, wherein R₈ is selected from the group consistingof boronic acid, boronate ion, arsenious acid, arsenite ion, telluricacid, tellurate ion, germanic acid, germanate ion, all optionallyprotected, and combinations thereof.
 21. The compound of claim 20,wherein each R₈ is an optionally protected boronic acid group.
 22. Thecompound of claim 19, wherein the compound comprises a fluorophore, andthe fluorescence of said fluorophore is modulated by the interaction ofsaid compound with glucose.
 23. The compound of claim 19, wherein R isan anthracene residue; R₁, R₂, R₃, R₄ and R₅ are hydrogen; R₆ and R₇ aredimethylamine residues; each R₈ is an optionally protected boronic acidgroup; one or both of R₉ and R₁₀ are aliphatic carboxylic acid residues;and each Z is carbon.
 24. The compound of claim 23, wherein one or bothof R₉ and R₁₀ are propionic acid residues.
 25. The compound of claim 1,wherein R is an anthracene residue; R₁, R₂, R₃, R₄ and R₅ are hydrogen;R₆ and R₇ are dimethylamine residues; each R₈ is an optionally protectedboronic acid group; R₉ and R₁₀ are the same or different and areselected from the group consisting of a methacrylamidoalkyl residue, amethacroyloxyethoxyalkyl residue, a hydroxyethoxyalkyl residue, and anaminoalkyl residue; and each Z is carbon.
 26. The compound of claim 19,wherein the compound is selected from the group consisting of:9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]-methyl]anthracene;9-[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[2-(carboxyethyl)amino]-methyl]anthracene;9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methylanthracene;9,10-bis[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methylanthracene;9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[5-aminopentylamino]-methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[6-(cyclohexanecarboxamido)hexylamino]methyl]anthracene;9-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[6-(cyclohexanecarboxamido)hexylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(carboxyethyl)amino]methyl]anthracene;9-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[2-(carboxyethyl)amino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[3-(N-6-(9-anthracenecarboxamido)hexylaminocarbonyl)ethylamino]methyl]anthracene;9-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(N-6-(9-anthracenecarboxamido)hexylaminocarbonylethylaminomethyl]anthracene;9-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[6-(3-carboxypropionamido)hexylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[6-(3-carboxypropionamido)hexylamino]methyl]anthracene;N-(3-Methacrylamidopropyl)-4-[2-N-[[2-(borono)benzyl]-[6-(N-[2-(borono)benzyl]-6-N-(3-carboxypropanamidoethyl)aminohexyl]]aminoethylamino]naphthalene-1,8-dicarboximide;N-Butyl-4-[2-N-[[2-(borono)benzyl]-[6-(N-[2-(borono)benzyl]-6-N-(2-methacrylamidoethyl)aminohexyl]]aminoethylamino]naphthalene-1,8-dicarboximide; and salts thereof.
 27. A detection system fordetecting the presence or concentration of glucose in a sample which mayalso contain an alpha-hydroxy acid or a beta-diketone, which comprises acompound having the following structure

wherein: R₁ and R₂ are the same or different and are selected from thefollowing: i) hydrogen; ii) a substituent to modify the pKa andhydrolytic stability of the R₈ moiety, iii) a detectable moiety, or iv)a linking group capable of attachment to a solid support or a polymericmatrix, said support or matrix optionally containing a detectablemoiety; R₃ is hydrogen or a linking group capable of attachment to asolid support or a polymeric matrix, said support or matrix optionallycontaining a detectable moiety; R₄ and R₅ are the same or different andare selected from the following: i) hydrogen, ii) a substituent tomodify the pKa and hydrolytic stability of the R₈ moiety, iii) adetectable moiety, or iv) a linking group capable of attachment to asolid support or a polymeric matrix, said support or matrix optionallycontaining a detectable moiety; each Z is independently carbon ornitrogen; R₆ and R₇ are the same or different and are i) linking groupshaving from zero to ten contiguous or branched carbon and/orheteroatoms, or ii) a linking group capable of attachment to a solidsupport or a polymeric matrix, said support or matrix optionallycontaining a detectable moiety; R is selected from the following: i) analiphatic and/or aromatic spacer containing from 1 to 10 contiguousatoms selected from the group consisting of carbon, oxygen, nitrogen,sulfur and phosphorus, ii) a detectable moiety, or iii) a linking groupcapable of attachment to a solid support or a polymeric matrix, saidsupport or matrix optionally containing a detectable moiety; each R₈ isthe same or different and is an optionally protected moiety which whenunprotected is capable of interaction with the vicinal diol groupspresent in glucose; and R₉ and R₁₀ are the same or different, and are i)hydrogen, ii) a detectable moiety, iii) a group which is a) a linkinggroup capable of attachment to a solid support or a polymeric matrix,said support or matrix optionally containing a detectable moiety, and/orb) includes a functional group capable of altering the physicalproperties of the compound; with the proviso that the indicator compoundcontains at least one detectable moiety associated therewith.
 28. Thedetection system of claim 27, wherein RB is selected from the groupconsisting of boronic acid, boronate ion, arsenious acid, arsenite ion,telluric acid, tellurate ion, germanic acid, germanate ion, alloptionally protected, and combinations thereof.
 29. The detection systemof claim 28, wherein each R₈ is an optionally protected boronic acidgroup.
 30. The detection system of claim 27, wherein the compoundcomprises a fluorophore, and the fluorescence of said fluorophore ismodulated by the interaction of said compound with glucose.
 31. Thedetection system of claim 27, wherein R is an anthracene residue; R₁,R₂, R₃, R₄ and R₅ are hydrogen; R₆ and R₇ are dimethylamine residues;each R₈ is an optionally protected boronic acid group; one or both of R₉and R₁₀ are aliphatic carboxylic acid residues; and each Z is carbon.32. The detection system of claim 31, wherein one or both of R₉ and R₁₀are propionic acid residues.
 33. The detection system of claim 27,wherein R is an anthracene residue; R₁, R₂, R₃, R₄ and R₅ are hydrogen;R₆ and R₇ are dimethylamine residues; each R₈ is a boronic acid group;R₉ and R₁₀ are the same or different and are selected from the groupconsisting of a methacrylamidoalkyl residue, a methacroyloxyethoxyalkylresidue, a hydroxyethoxyalkyl residue, and an aminoalkyl residue; andeach Z is carbon.
 34. The detection system of claim 27, wherein thecompound is selected from the group consisting of:9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]-methyl]anthracene;9-[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[2-(carboxyethyl)amino]-methyl]anthracene;9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methylanthracene;9,10-bis[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methylanthracene;9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[5-aminopentylamino]-methyl]anthracene;and 9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[6-(cyclohexanecarboxamido)hexylamino]methyl]anthracene;9-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[6-(cyclohexanecarboxamido)hexylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(carboxyethyl)amino]methyl]anthracene;9-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[2-(carboxyethyl)amino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[3-(N-6-(9-anthracenecarboxamido)hexylaminocarbonyl)ethylamino]methyl]anthracene; 9-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(N-6-(9-anthracenecarboxamido)hexylaminocarbonylethylaminomethyl]anthracene; 9-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolano)benzyl]-N-[6-(3-carboxypropionamido)hexylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[6-(3-carboxypropionamido)hexylamino]methyl]anthracene;N-(3-Methacrylamidopropyl)-4-[2-N-[[2-(borono)benzyl]-[6-(N-[2-(borono)benzyl]-6-N-(3-carboxypropanamidoethyl)aminohexyl]]aminoethylamino]naphthalene-1,8-dicarboximide;N-Butyl-4-[2-N-[[2-(borono)benzyl]-[6-(N-[2-(borono)benzyl]-6-N-(2-methacrylamidoethyl)aminohexyl]]aminoethylamino]naphthalene-1,8-dicarboximide; and salts thereof.